Mutiple gene expression for engineering novel pathways and hyperexpression of foreign proteins in plants

ABSTRACT

Introducing blocks of foreign genes in a single operon would avoid complications such as position effect and gene silencing inherent in putting one gene at a time into random locations in the nuclear genome. Cloning several genes into a single T-DNA does not avoid the compounded variable expression problem encountered in nuclear transgenic plants. This disclosure shows that a bacterial operon can be expressed in a single integration event as opposed to multiple events requiring several years to accomplish. Expression of multiple genes via a single transformation event opens the possibility of expressing foreign pathways or pharmaceutical proteins involving multiple genes. Expressing the Cry2aA2 operon, including a putative chaperonin to aid in protein folding, in the chloroplast via a single transformation event leads to production of crystalized insecticidal proteins. Expressing the Mer operon via a single transformation event leads to a phytoremediation system.

RELATED APPLICATIONS

[0001] This patent application claims the benefit of U.S. ProvisionalApplications Nos. 60/185,660, filed Feb. 29, 2000, 60/257,408, filedDec. 22, 2000, 60/259,248 filed Dec. 29, 2000 and 60/266,121 filed Feb.2, 2001. All applications are here incorporated by reference.

STATEMENT REGARDING FEDERALLY SPONSORED FEDERAL RESEARCH

[0002] The work of this invention is supported in part by theUSDA-NRICGP grants 95-82770, 97-35504 and 98-0185 to Henry Daniell.

FIELD OF THE INVENTION

[0003] This application pertains to the field of genetic engineering ofplant genomes, particularly plastids and to methods of and engineeredplants with operons that lead to and result in overexpression of thegene of interest. This application also pertains to the field of geneticengineering of algal and bacterial genomes.

DESCRIPTION OF RELATED ART

[0004] Karamata, in U.S. Pat. No. 4,797,279, proposed the generation ofBacillus thuringiensis hybrids that have insecticidal properties throughconjugation. Conjugation is mediated by a conjugative plasmid functionalin the B.t. kurstaki strain and the B.t. tenebrionis strain. Theresulting hybrid is capable of producing each of the delta-endotoxincrystals typical for a B.t. kurstaki strain and a B.t. tenebrionisstrain.

[0005] McBride, in U.S. Pat. No. 5,545,818 and McBride et al. (1995),describes a method of genetically engineering the plastids of a plant orplant cell such they provide increased expression of the Bacillusthuringiensis insecticidal proteins in the plastids. A constructcontaining a promoter functional in plant plastids, a single geneencoding an insecticidal Bacillus thuringiensis toxin, another DNAsequence encoding a selectable marker, and a transcription terminationregion capable of terminating transcription in a plant plastid, is usedto affect plant transformation. The transcription and translation of theB.t gene product occurs in the plastids.

[0006] Daniell et. al., in U.S. Pat. No. 5,932,479 (1999), entitled“Genetic engineering of plant chloroplast,” teaches plant cellschloroplast transformed by means of an expression cassette comprising anexogenous DNA sequence which is stably integrated to the chloroplastgenome of the cell of a target plant. “Stably” integrated DNA sequencesare those which are inherited through genome replication by daughtercells or organisms. This stablility is exhibited by the ability toestablish permanent cell lines, clones, or transgenic plants comprisedof a population containing the exogenous DNA.

[0007] Likewise, U.S. Pat. No. 5,693,507 (1997) to Daniell and McFaddendiscloses such stable integration of the chloroplast by means of anexpression cassette which comprises an exogenous DNA sequence whichcodes for a desired trait, and the transformed plant.

[0008] Daniell, in PCT International Publication WO 99/10513, teachesthe composition and use of universal chloroplast integration andexpression by vectors to stably transform and integrate genes ofinterest into chloroplast genome of multiple species of plants. Thisleads to chloroplast expression of genes of interest. Transformed plantsshow the highest level of expression. Plants transformed withinsecticidal genes are lethal to insects that are 40,000-fold resistantto Bt. insecticidal proteins.

[0009] Significantly, in the prior art inventions use multiple promotersto drive the expression of multiple genes. Put differently, theinventions of the prior art employ a single promoter to drive a singlemonocistron. In contrast, the present invention employs a singlepromoter to drive polycistrons, resulting in equal levels of expressionof the polycistrons.

[0010] All publications and patents are hereby incorporated byreference.

BACKGROUND OF THE INVENTION

[0011] In plant and animal cells, nuclear mRNAs are translatedmonocistronically. This poses a serious problem when engineeringmultiple genes in plants. Therefore, in order to express thepolyhydroxybutyrate polymer or Guy's 13 antibody, single genes werefirst introduced into individual transgenic plants, then these plantswere back-crossed to reconstitute the entire pathway or the completeprotein. Similarly, in a seven year-long effort, Ye et al. recentlyintroduced a set of three genes for a short biosynthetic pathway thatresulted in β-carotene expression in rice. ID contrast, most chloroplastgenes of higher plants are co-tanscribed. Multiple steps of chloroplastmRNA processing are involved in the formation of mature mRNAs.

[0012] In accordance of the invention expression of polycistrons via theplastid genome, in particular the chloroplast genome, provides a uniqueopportunity to express entire pathways in a single transformation event.Additionally, chloroplast genetic engineering is an environmentallyfriendly approach resulting in containment of foreign genes andhyper-expression.

[0013] Plant bioremediation (phytoremediation), is the use of plants forin-situ restoration of contaminated sites. The technique has risen inthe last decade as a strong and safe technique to address with theincreasing problems of the pollution of soil and water bodies. In thepast other techniques, such as mechanical and bacterial bioremediationwere implemented with little success, since they were costly andthreatened the safety of our environment. Plants, on the other hand, areadvantageous for bioremediation systems since they have a high capacityfor adaptation to different environments and a natural resistanceagainst different toxic pollutants. They are cheap, non-evasive and helpcontain disrupted ecosystems. These characteristics make plants an idealvehicle for bioremediation.

[0014] Mercury is a toxic heavy metal that is commonly released into theenvironment as a byproduct of different chemical reactions of modernindustries. The present world production of mercury is about 9000tons/year (http://www.chem.ualberta.cal.htm). In the environment,mercury is rapidly methylated by methanogenic bacteria (Ex Desulfovibriodesulfuricans) producing the 10 fold more toxic organomercurials(Compeau et. al.) 1985; Gilmour et al. 1992). Organomercurials are moretoxic due to its increased hydrophobicity, which allows it to crosslipid membranes because it is more hydrophobic than mercury. Over 90% ofthe intake of methylmercury is absorbed into blood compared with only 2%of inorganic mercury (http://www.chem.ualberta.cal.htm). Bothorganomercurials and mercury have the tendency to accumulate in thetissue, especially in the membrane bound organelles. In plants organicmercury crosses the lipid membrane of organelles, for examplechloroplast, where it can poison essential oxidative and photosyntheticelectron transport chains more easily than metallic mercury (Rugh et.al. 1996). In photosynthetic organisms, mercury affects theoxygen-evolving complex that is found in the photosystem II and is boundto the thylakoid membrane (Bernier et al. 1993). Mercury treatment ofPSII leads to a strong inhibition of oxygen evolution by removal of EP33(one of the proteins of the OEE complex; Bernier et al. 1995). Mercuryreduces the Fm and Fv values due to additional inhibitory sites on thedonor side of PSII, including damage to the light-photochemistry (Rashidet al. 1990). Medical researchers discovered that high levels ofmethylmercury cause severe neurological degeneration in birds, cats andhumans (Minamata Disease Research Group, 1968; Harada et al. 1995).Thus, mercury and organomercurials are ideal targets forphytoremediation.

[0015] In water, mercury pollution also poses a problem. Mercuryaccumulates in the sediments of lakes and oceans where methanogenicbacteria live (http://ehpnet.niehs.nih.gov). These bacteria methylatemercury to produce methylmercury, which is eventually released intowater (Harada et al. 1995). The methylmercury is trapped into the smallfish when the water passes through their gills or they feed onphytoplanktons that carry high concentrations of the pollutant.Predatory fish, as bass in fresh water and tuna in salt water, live forlong periods of time feeding on smaller fish. During their life span,they can accumulate high levels of methylmercury that can reach 1.0 ppmin normal water and 30 ppm in areas of high pollution with mercury(http://ehpnet.niehs.nih.gov) Then, humans and birds feed oncontaminated fish and accumulation in their tissue cause severeneurological damage.

[0016] Meagher and colleagues have used a nuclear modified form of themerA and merB genes to transform plants that are resistant to mercuryand organomercurials respectively (Bizily et al. 1999; Ruga et. al.1996), U.S. Pat. No. 5,965,796 (1999). One of the drawbacks of nucleargenetic engineerig is that it requires several back crosses to createthe complete pathway that detoxifies mercury and organomercurials(Bizily et al. 2000). This results in variation in expression levelsamong different transgenic lines and tolerance to differentconcentrations of organomercurials, only in low levels of tolerance (10μM) (Bizily et al. 2000). Another concern of the use of nucleartransformed plants in-situ is the escape of the foreign genes via pollen(Daniell 1999; Bogorad, 2000).

[0017] The present invention provides a transgenic plant bioremediationsystem for soil as well as a transgenic algae/bacteria bioremediationsystem for water.

Non-obviousness of Expression of Operons Via the Chloroplast Genome

[0018] Despite the potential advantages of chloroplasts for foreign geneexpression, it was not obvious that multiple genes expressed by a singlepromoter in chloroplasts would be expressed in this organelle in acoordinated manner. Polycistrons have been observed in chloroplasts inthe past but processing RNA sequences present in between individualtranscripts, proteins or enzymes involved in processing or cofactorsnecessary for processing of polycistrons have not yet been characterizedTherefore, it was not obvious to one skilled in the art that multipleforeign gene transcripts would be properly processed and translated whenexpressed from a heterologous promoter.

[0019] Prior to this patent application there were no published reportsof expression of multiple genes in chloroplasts and there were validreasons to suggest that it would be problematic. Indeed, despite severalreports of foreign gene expression via the chloroplast genome, no oneever attempted expression of a bacterial operon via the chloroplastgenome because of inadequate understanding of processing of polycistronswithin plastids. All foreign genes engineered via the plastid genomehave been driven by individual promoters and 3′ regulatory sequences. Itwas not known whether 3′ terminators and regulatory sequences werenecessary for individual genes of the foreign operon. It is generallybelieved that the proteins or enzymes involved in processing may beunder the control of the nuclear genome. It was also believed that theremaybe several environmental factors involved in processing polycistrons,including light.

[0020] While chloroplast ribosome binding sites have been characterized,it was not obvious that ribosome binding sites or untranslated regionsupstream of bacterial genes would function in plastids. Also, it was notanticipated that a chaperonin present in a bacterial cell would functionwithin chloroplasts and help fold the foreign protein or interfere withfolding of other chloroplast proteins. It was certainly unanticipatedthat it was possible to create cuboidal crystals within chloroplastsduplicating the functions of a bacterium during sporulation or duplicatebioremediation pathways within plastids. There was no certainty that theenzymes of the pathway or proteins of the operon would be expressed in acoordinated manner.

[0021] Indeed, the prior art suggested that there might have beenunforeseen deleterious effects of high-level expression of severalforeign proteins within chloroplasts on plant growth or development thatwere not apparent from the experiences with other transgenes. The pH andoxidation state of the chloroplast differs from that of bacterial cellsin ways that might inhibit or prevent functions of proteins or enzymes.Because the results of this invention contradicted those teachings ofthe prior art, this invention was characterized as breakthrough in plantbiotechnology and featured on the cover of Nature Biotechnology (themost prestigious biotechnology journal in the world) in January 2000.Scientists around the world have written reviews subsequent to thatpublication appreciating this invention. Engineering multiple genes intransgenic plants via the nuclear genome is not only extremely timeconsuming (taking several years to accomplish) but is riddled withproblems of position effect, gene silencing etc. Therefore, thisaccomplishment was characterized as the holy-grail of plantbiotechnology.

SUMMARY OF THE INVENTION

[0022] By this invention, plastid expression constructs are providedwhich are useful for genetic engineering of plant cells and whichprovide for enhanced expression of several foreign proteins in plastidsutilizing a single transformation event. The transformed plastid ispreferrably a metabolically active plastid, such as the chloroplastsfound in green and non-green plant tissues including leaves and otherparts of the plant. This invention opens the door to engineering novelpathways for metabolic engineering and gene stacking, or for multisubunit complex proteins requiring stoichiometric and coordinatedexpression of multiple genes. The plastid is preferably one which ismaintained at a high copy number in the plant tissue of interest.

[0023] The present invention is applicable to all plastids of plants.These include chromoplasts which are present in the fruits, vegetablesand flowers; amyloplasts which are present in tubers like the potato;proplastids in roots; leucoplasts and etioplasts, both of which arepresent in non-green parts of plants.

[0024] The plastid expression constructs for use in this inventiongenerally include a single plastid promoter region and multiple genes ofinterest to be expressed in transformed plastids. The DNA sequence ofinterest may contain a number of consecutive encoding regions, to beexpressed as an operon, for example where introduction of a foreignbiochemical pathway into plastids is desired for metabolic engineeringor gene stacking. Plastid expression constructs of this invention islinked to a construct having a DNA sequence encoding a selectable markerwhich can be expressed in a plant plastid.

[0025] In a preferred embodiment, transformation vectors for transfer ofthe construct into a plant cell include means for inserting theexpression and selection constructs into the plastid genome. Thispreferably comprises regions of homology to the target plastid genomewhich flank the constructs.

[0026] The chloroplast vector or constructs of the invention preferablyinclude a universal chloroplast expression vector which is capable ofimporting a desired trait to a target plant species. Such a vector iscompetent for stably transforming the chloroplast genome of differentplant species which comprises an expression cassette which is describedfurther herein. Such a vector generally includes a plastid promoterregion operative in said plant cells chloroplast, a gene which is linkedto a multi-gene operon which includes an ORF which codes for a putativechaperonin which facilitates the folding of the protein to formproteolytically stable cuboidal crystals. Preferably, one or more DNAsequences of interest to be expressed in the transformed plastids.

[0027] The invention provides also a plastid vector comprising of a DNAconstruct. The DNA construct includes a 5′ part of a plastid DNAsequence inclusive of a spacer sequence; a promoter that is operative inthe plastid; at least a heterologous DNA sequence encoding multiplepeptides of interest; a gene that confers resistance to a selectablemarker; a multi-gene operon; a transcription termination regionfunctional in the target plant cells; and a 3′ part of the plastid DNAsequence inclusive of a spacer sequence. The DNA construct is flanked byDNA sequences which are homologous to the spacer sequence of the targetplastid genome. The plastid is preferably a chloroplast. The vectorpreferably includes a ribosome binding site and a 5′ untranslated region(5′UTR). A promoter operative in the green and non-green plastids is tobe used in conjunction with the 5′UTR,

[0028] The invention provides a promoter that is operative in the greenand non-green plastids of the target plant cells such as the psbApromoter, rbcL promoter, atpβ promoter region, accD promoter, and the16SrRNA promoter.

[0029] The invention provides a gene, which can be a mutant gene, thatconfers resistance, such as antibiotic resistance, to a selectablemarker like the aadA gene.

[0030] The invention provides a cassette which can be modified toinclude a selectable marker, a gene encoding the chaperonin and anydesired heterologous gene. Such applications will be beneficial for thehigh level production in plants of other desired protein products aswell

[0031] Further, the invention preferably provides a three-geneinsecticidal Bacillus thuringiensis (Bt) operon which shows operonexpression and crystal formation via the chloroplast genome. The operoncomprises of three operably linked components which operate in concertas a biosynthetic pathway: a distal gene which codes for a insecticidalprotein and two open reading frames (ORF). The two ORFs code for atleast one molecule of interest and at least one chaperonin to assist inthe folding of the insecticidal protein. The molecule of interest ofthis operon can be a peptide, an enzyme, a selectable marker, or abio-pharmaceutical, including monoclonals.

[0032] This invention also provides for other three-gene operons,particularly insecticidal operon or the Cry2Aa2 operon. This inventionalso provides for operons of the Cry or Cyt series.

[0033] An operon of this invention further provides that the second ORF(ORF2) codes for a putative chaperonin. A chaperonin is a molecule whichfacilitates the folding and assembly of proteins to form functionalproteolytically stable cuboidal crystals. The ORF2 is operably linked toa gene encoding the insecticidal protein. The invention provides abacterial chaperonin that is capable of facilitating the folding andassembly of insecticidal proteins.

[0034] This invention also provides crystalized insecticidal proteinssuch as δ-endotoxin proteins, Cry proteins such as the Cry2Aa2 proteins,or Cyt proteins.

[0035] In accordance of the invention, the introduction blocks offoreign genes in a single operon would avoid complications inherent innuclear transformation such as position effect and gene silencing inputting one gene at a time into random locations in the nuclear genome.Repeated use of a single promoter causes gene silencing (De Wilte, C.et. al. 2000). Cloning several genes into a single T-DNA does not avoidthe compounded variable expression problem encountered in nucleartransgenic plants. This invention shows that a bacterial operon can beexpressed in a single integration event. Expression of multiple genesvia a single transformation event opens the possibility of expressingforeign pathways or pharmaceutical proteins involving multiple genes.

[0036] The invention provides for the demonstration of expression of abacterial operon or polycistrons in transgenic plants and opens the doorto engineer novel pathways in plants in a single transformation event.

[0037] The invention provides a single vector or construct (or cassette)which encodes more than one heterologous protein product. Thisembodiment of the invention provides that a heterologous DNA fragmentthat is introduced into a universal vector encodes more than one gene.In one example shown, this invention discloses, the DNA encodes anoperon of three genes and produces proteins from at least two genes, oneof those genes encode a protein and a chaperone protein. This aspect ofthe invention—to co-expressing multiple genes—is beneficial if oneskilled in the art desires to introduce a biosynthetic pathway thatcomprises multiple steps into plants. For example, a three stepsynthesis of a desired compound might require three different enzymes. Asingle transformation will generate a recombinant plant possessing allthree heterologous enzymes which can function in concert to produce thedesired product.

[0038] Thus another embodiment of the invention relates to the maximalproduction of a heterologous protein by co-expressing it with anotherpolypeptide that induces crystallization of said protein. This aspect ofthe invention provides the yield of heterologous gene expression isgreatly enhanced if the desired protein is in crystal form in thetransformed plant. The increased yield is because the crystal form ofthe proteins protected them from cellular proteases. This isaccomplished by co-expressing the desired gene with a second geneencoding a chaperonin that directs crystallization.

[0039] Also, formation of crystals of foreign proteins opens a simplemethod of purification via centrifugation. Plants transformed with thecry2Aa2 operon of the invention show a large accumulation and improvedpersistence of the expressed insecticidal protein(s) throughout the lifeof the plant. This is most likely because of the folding of theinsecticidal protein into cuboidal crystals, there by protecting it fromproteases. This is an environmentally friendly approach because foldedcrystals improve the safety of the Bt transgenic plants. In contrast tocurrently marketed transgenic plants that contain soluble CRY proteins,folded protoxin crystals will be processed only by those target insectsthat have high alkaline gut environment In addition, absence ofinsecticidal protein in transgenic pollen eliminates toxicity tonon-target insects via pollen. Expression of the cry2Aa2 operon inchloroplasts provides a model system for hyper-expression of foreignproteins in a folded configuration enhancing their stability andfacilitating single step purification. This is the first successfuldemonstration of expression of a bacterial operon in transgenicchloroplast plants.

[0040] The invention provides a model system for large-scale productionof foreign protein within chloroplasts in the folded configurationenhancing their stability and facilitating single-step purification, forexample, biopharmaceuticals such as human serum albumin (HSA) andinsulin.

[0041] All known methods of transformation can be used to introduce thevectors of this invention into target plant plastids includingbombardment, PEG Treatment, Agrobacterium, microinjection, etc.

[0042] The invention provides transformed crops, like solanaceous plants(monocotyledonous and dicotyledonous). Preferably, the plants are ediblefor mammals, including humans.

[0043] The invention provides target Bt transgenic plants which arelikely to show a more stable protein expressed at high levels in thechloroplast throughout the growing season. It should increase toxicityof Bt transgenic plants to target insects and help eliminate thedevelopment of Bt resistance. The invention provides an example of thecry2Aa2 bacterial operon is expressed in tobacco chloroplasts to testthe resultant transgenic plants for increased expression and improvedpersistence of the accumulated insecticidal protein(s).

[0044] The invention provides transformed plants including leaves whichaccumulated a high percent of total soluble protein (close to 50%) inmature leaves and remain stable even in old bleached leaves.

[0045] The invention provides transformed plants which are resistant todifficult-to-control insects, like cotton boll worm, which were killed100% after consuming transgenic leaves. The invention also providesplants which contain insecticidal protein fold into cuboidal crystals.Plants which contain protoxin crystals, which will be processed only bytarget insects that have high alkaline gut environment, which shouldimprove safety of Bt transgenic plants. Also, plants are free ofinsecticidal proteins in transgenic pollen, which eliminates toxicity tonon-target insects via pollen such as Monarch butterfly larvae. Theinvention provides electron microscopic proof of the presence of thecuboidal crystals inside the chloroplast.

[0046] The invention also provides an environment friendly approach toengineering insect resistance to plants because folded crystal productsimprove the safety of the Bt transgenic plants which will be edibleconsume, mammals, including humans.

[0047] In another embodiment of the invention provides heterologous DNAsequences which mediate resistance to (a) heavy metal in transgenicplants or plant cells which express these coding sequences encodingmetal ion reductases and (b) organomercurial compounds in transgenicplants or plant cells which express the coding sequence encodingorganomercury lyase. Preferably the coding sequence is that of merA(which encodes mercuric ion reductase) and merB (which encodesorganomercury lyase).

[0048] The present invention provides a chloroplast universal vectorwhich contains a Mer operon containing metal resistance coding sequencesoperably linked to transcriptional and translational control sequenceswhich are functional in the chloroplast in target plants. Preferably thecoding sequence is that of merA and merB. Also, the present inventionprovides transgenic plant cells, plant tissue and plants whosechloroplast has been modified to contain and express two metalresistance coding sequences operably linked to transcriptional andtranslational control sequences which are functional in the chloroplastof target plants. Preferably the coding sequences are that of merA andmerB. Also provided by the present invention are methods for effectingmetal resistance inplants by stably transforming a plant to contain andexpress two heterologous DNA sequences encoding metal resistanceoperably linked to transcriptional and translational control sequenceswhich are functional in the chloroplasts of target plants. Preferablythe coding sequence is that of merA and merB.

[0049] The present invention are methods for effecting metal resistancein plants by stably transforming a green algae or cyanobacteria tocontain and express two heterologous DNA sequences encoding metalresistance operably linked to transcriptional and translational controlsequences which are functional in the target green algae orcyanobacteria. Preferably the coding sequence is that of merA and merB.

[0050] A further object of the invention are transgenic plants, algaeand bacteria which contain and express metal resistance andorganomercurial compound resistance coding sequences.

[0051] Other embodiments of the invention are described in greaterdetail hereinafter.

BRIEF DESCRIPTION OF THE DRAWINGS

[0052]FIG. 1 shows (a) Chloroplast expression vector pLD-BD Cry2Aa2operon (9.8 kb) with PCR primer landing sites and expected fragmentsizes. PCR analysis of untransformed and putative chloroplasttransformants using two primer sets: (b) 1P1M; and (c) 3P3MN Lane 1: 1kb ladder; Lane 2: untransformed; Lanes 3-7: pLD-BD Cry2Aa2 operonputative transformants; Lane 8: pLD-BD Cry2Aa2operon plasmid DNA.

[0053]FIG. 2 shows the Southern blot analysis of T₀ and T₁ generations.A. The 0.81 kb probe BamH1-Bglll fragment containing the chloroplastflanking sequence. Untransformed plants generate a 4.47 kb fragment. B.Vector map showing Cry2Aa2 operon transformed 32P labeled fragments of8.42 kb and 1.42 kb. C. Lane 1: 1 kb ladder; Lane 2: untransformed;Lanes 3-7; T0 transgenic lines; Lanes 8-9: T1 transgenic lines.

[0054]FIG. 3 shows the 10% SDS-PAGE gel stained with R-250 CoomassieBlue. Loaded protein concentrations are provided in parenthesis. Lanes1: prestained protein standard; Lane 2: partially purified Cry2Aa2protein from E. Coli (5 μg); Lane 3: Single Gene derived Cry2Aa2 pelletextract solubilized in 50 mM NaOH (22.4 μg); Lane 4: Single Gene derivedCry2Aa2 supernatant (66.5 μg); Lane 5: Operon Derived Cry2Aa2supernatant (58.6 μg); Lane 7: untransformed tobacco pellet extractsolubilized in 50 mM NaOH (29.8 μg); Lane 8: untransformed tobaccosupernatant (30.4 μg). Colored compounds observed in the supernatant oftransgenic plants interfered with the DC Bio-Rad protein assays but notin the pellet.

[0055]FIG. 4 shows the quantification of Single Gene derived Cry2Aa2 andOperon Derived Cry2Aa2 proteins by ELISA as a percentage of totalsoluble protein in young, mature, and old transgenic leaves. A: SingleGene derived Cry2Aa2 expression shown as a percentage of total solubleprotein. B: Operon derived Cry2Aa2 expression shown as a percentage oftotal soluble protein.

[0056]FIG. 5 shows the insect bioassays of untransformed tobacco leaves(A,D,G), Single Gene derived Cry2Aa2 transformed leaves (B,E,H) andOperon Derived Cry2Aa2 transformed leaves (C,F,I). A, B, C: bioassayswith H. virescens; D, E, F: bioassays with H. zea; G, H, I: bioassayswith S. exigua.

[0057]FIG. 6 shows the electron micrographs of Operon Derived Cry2Aa2leaf sections in young (A), mature (BD) and old, bleached leaf (C).Single Gene derived Cry2Aa2 mature leaf (E), mature untransformed leaf(F).

[0058]FIG. 7 shows the phenotypes of untransformed (A) or transformedwith the cry2Aa2 gene (B) or cry2Aa2 operon (C).

[0059]FIG. 8 shows a pLD-merAB chloroplast vector

[0060]FIG. 9 shows transformed E. Coli grown in 100 μm HgCl₂.Transformed E. coli cells containing the vectors pLD-merAB andpLD-MerAB-3′UTR grown in LB at different concentrations of HgCl₂. Platesshow transformed cells growing at 100 μM HgCl₂. No growth was observedin the control.

[0061]FIG. 10 shows chloroplast transgenic plants. A: Transgenic plantshoot induction in RMOP with 500 μg/ml Spec. B: Transgenic plant rootinduction in MSO with 500 μg/ml Spec. C: Transgenic plant grown in soil.

[0062]FIG. 11 shows integration of the mer operon into the chloroplastgenome. A: PCR using specific primers that land in the gene cassette(5P/2M) show a product of 3.8 kb size (clones2, 4, 5, 7, 9, 11). Clones1 and 3 show no integration of the cassette. Positive control, isplasmid pLD-merAB-3′UTR. Negative control is untransformed plant DNA. B:PCR using specific primers that land within the native chloroplastgenome (3P/3M), eliminate mutants (clone 3), showing integration of thecassette into the chloroplast genome (clones: 1, 2, 4, 5, 6, 7,9, 11.1.6 kb PCR product).

[0063]FIG. 12 shows the Chlorella vulgaris vector construct.

[0064]FIG. 13 shows the Synechocystis vector construct.

[0065]FIG. 14 shows the Lemna vector construct.

[0066]FIG. 15 shows the Sugarcane vector construct.

[0067]FIG. 16 shows confirmation of Lemna vector construct.

[0068]FIG. 17 shows confirmation of Sugarcane vector construct.

[0069]FIG. 18 shows other vectors suitable for operon expression.

DETAILED DESCRIPTION OF THE INVENTION

[0070] This invention is related to transformation of the plastid genomeapplicable to all plastids of plants. These include chromoplasts whichare present in the fruits, vegetables and flowers; amyloplasts which arepresent in tubers like the potato; proplastids in roots; leucoplasts andetioplasts, both of which are present in non-green parts of plants

[0071] The invention provides in one aspect a single vector or constructwhich encodes more than one heterologous protein product. The secondaspect of the invention relates to the maximal production of aheterologous protein by compressing it with another polypeptide or achaperone that induces crystallization of said protein.

[0072] The first aspect of the invention provides that a heterologousDNA fragment that is introduced into a plastid vector (described below)encodes more than one gene. In one example, the DNA encodes an operon ofthree genes and produces proteins from three genes. This aspect of theinvention to co-expressing multiple genes is beneficial if one desiresto introduce a biosynthetic pathway into plants that comprises multiplesteps. For example, a three step synthesis of a desired compound mightrequire three different enzymes. Co-expressing all three enzymes in thechloroplast can be accomplished according to this invention. Thus, asingle transformation will generate a recombinant plant possessing allthree heterologous enzymes which can function in concert to produce thedesired product.

[0073] The second aspect of the invention is that the yield ofheterologous gene expression is greatly enhanced if the desired proteinis in crystal form in the transformed plant, as provided by thisinvention. This is accomplished by coexpressing the desired gene with asecond gene encoding a chaperone protein that directs crystallization.Data given in the specification shows almost 100 fold greater amounts ofinsecticidal protein can be found in plants co-expressing the chaperoneprotein versus plants having only the gene encoding the insecticidalprotein. The expression cassette itself can be modified to include aselectable marker, a gene encoding the chaperone protein and any desiredheterologous gene. Such applications will be beneficial for the highlevel production in plants of other desired protein products as well.

[0074] A flyer aspect of the invention describes a plant bioremediationsystem. A plant bioremediation system employing chloroplasttransformants have a number of advantages. First, plants have thegenetic capacity (using hundreds, even thousands, of genes) to extractat least 16 metal cation and oxyanion nutrients from the soil and groundwater. This capacity can be chemically and genetically manipulated toextract environmental pollutants. Second, plants have extensive rootsystems to help in this mining effort; typical estimates are as high as100×10⁶ miles of roots per acre [Dittner, H. J. (1937) Amer. J. Botany24:417-420]. The root systems of various macrophytes can reach up to 40feet into the soil. In addition, plants are photosynthetic and govern asmuch as 80% of the available energy at any given time in mostecosystems. Through photosystem I (a system not found in photosyntheticbacteria), they use light energy to generate large amounts of reducingpower (as NADPH) that can be used to efficiently reduce metal ions.Plants photosynthetically fix CO₂ and reduce it to make their owncarbon/energy source. This reduced carbon energy is used by plant rootsto live heterotrophically. This redox power can also be used to reducetoxic metal ions like Hg(II) [Rugh et al. (1996) supra]. Many plants canproduce large amounts of biomass annually with the potential both toenrich contaminated soil with carbon and nutrients and/or remove metalions from the soil. The site of action of mercury within thechloroplasts, ability to express bacterial operon via the chloroplastgenome, and several other environmental benefits of chloroplast geneticengineering make this an advantageous system for metal remediation.

[0075] An additional benefit of the metal resistant plants is theirability to harvest metals; precious and semi-precious metals can bereduced and thereby trapped in plant tissues. These metals include caninclude gold, silver, platinum, rhenium, copper, palladium, nickel, zincand cadmium, where the corresponding metal ions are reduced by the metalresistance gene product in those plants.

[0076] In addition, this invention also introduces a novel approach formercury and organomercurial bioremediation in water. Two organisms areused as model systems. One is Synechocystis, a photosynthetic bacterium(Cyanobacterium) that grows in salt and fresh water (in a hightemperature range, from ice to hot springs). The other is Chlorellavulgaris, a green algae that grows in fresh water. These organisms aretransformed with the merA and merB genes (mer operon) to remove mercuryand organomercurials from water. Transformed cells could be applied forsludge treatment and in water treatment to remove organomercury andmercury form water and sediments before releasing them to theenvironment, especially from industrial effluents that generatebyproducts with mercury.

[0077] “Metal resistance” means that a non-naturally occurring organismis not inhibited by the presence of at least one of divalent cations ofmercury, cadmium, cobalt, trivalent cations of gold, and monovalentsilver ion, at concentrations (levels) at which a naturally occurring(wild-type) counterpart of the non-naturally occurring organism isinhibited or exhibits symptoms of toxicity. It is not intended that theterm metal resistance refer to resistance to unlimited concentration ofmetal ions, but rather the term is relative in that it relies oncomparison to the properties of a parental strain.

[0078] A “metal resistance coding sequence” is one which encodes aprotein capable of mediating resistance to at least one metal ion,including, but not limited to, divalent cations of mercury, nickel,cobalt, trivalent cations of gold, and by monovalent cations of silver.Also within the scope of this definition are mutant sequences whichdetermine proteins capable of mediating resistance to divalent cationsof lead, cadmium and copper.

[0079] An “organomercurial resistance coding sequence” is one whoseprotein product mediates resistance to such organic mercury compounds asalkylmercurials and certain aromatic mercurials, for example, mono- ordimethylmercury, typically in conjunction with a metal resistance genesuch as merA. As specifically exemplified herein, the organomercurialresistance gene is the methylmercury lyase gene (merB) and its geneproduct confers resistance to organomercurial compounds such asmethymercury, p-chloromercuribenzoate (PCMB) andp-hydroxymercuribenzoate in conjunction with the merA gene product(mercury ion reductase).

[0080] The metal resistance protein (MerA protein, mercuric ionreductase) is exemplified by that from Tn21, a bacterial mercuryresistance transposon originally isolated from the IncFII plasmid NR1.In addition to reducing mercuric ions, the Tn21 MerA reduces trivalentgold and monovalent silver cations [Summers and Sugarman (1974) Journalof Bacteriology 119:242-249]. Monovalent silver and certain divalentmetal cations have been shown to be competitive inhibitors of mercuricion reduction in vitro [Rinderle et al. (1983) Biochemistry 22:869-876].MerA mediates resistance to trivalent gold, divalent cobalt divalentcopper and divalent nickel cations as well as divalent ionic mercury.

[0081] It is understood that nucleic acid sequences from nucleotide 14through nucleotide 1708, or MerApe 20, MerApe 29, MerApe 38 or MerApe 47will function as coding sequences synonymous with the exemplifiedmerApe9 coding sequence. Nucleic acid sequences are synonymous if theamino acid sequences encoded by those nucleic acid sequences are thesame. The degeneracy of the genetic code is well known to the art; i.e.,for many amino acids, there is more than one nucleotide triplet whichserves as the codon for the amino acid; for expression in plant cells ortissue it is desired that codon usage reflect that of plant genes andthat CpG dinucleotides be kept low in frequency in the coding sequence.It is also well known in the biological arts that certain amino acidsubstitutions can be made in protein sequences without affecting thefunction of the protein. Generally, conservative amino acidsubstitutions or substitutions of similar amino acids are toleratedwithout affecting protein function. Similar amino acids can be thosethat are similar in size and/or charge properties, for example,aspartate and glutamate and isoleucine and valine are both pairs ofsimilar amino acids. Similarity between amino acid pairs has beenassessed in the art in a number of ways. For example, Dayhoff et al.(1978) in Atlas of Protein Sequence and Structure, Vol. 5, Suppl. 3, pp.345-352, which is incorporated by reference herein, provides frequencytables for amino acid substitutions which can be employed as a measureof amino acid similarity. Dayhoff et al.'s frequency tables are based oncomparisons of amino acid sequences for proteins having the samefunction from a variety of evolutionarily different sources.

[0082] The expression of merB in plants confers resistance to and/or theability to detoxify organomercurials including, but not limited to,alkylmercury compounds wherein the alkyl group is either straight chainor branched, alkenyl mercury compounds, allyl mercury, alkynyl mercurycompounds, aromatic mercury compounds, wherein there are from one toabout 6 aromatic rings, and other organomercurials including but notlimited to humic acid-containing mercury compounds. The MerB proteinalso mediates resistance to and/or detoxifies organo-metals including,but not limited to, organic lead, organic cadmium and organic arseniccompounds, where those organometals can be alkyl, aklenyl, alkynyl oraromatic metal compounds.

[0083] Coding sequences suitable for expression in a plant are operablylinked downstream of a constitutive or a regulated promoter construct.Transgenic plants can be constructed by use of chloroplast universalvector containing a 5′ a part of a chloroplast spacer sequence, apromoter that is operative in the chloroplast of the target plant cells,at least two heterologous DNA sequences encoding merA and merB, a genethat confers resistance to a selectable marker, a transcriptiontermination region functional in the target plant cells; and a 3′ partof the chloroplast spacer sequence. Alternatively, the vector may notcontain a terminator.

[0084] The mer operon-expressing plants can be used in the remediationof mercury-contaminated soil to block the biomagnification of methylmercury up the food chain. Deep-rooted trees like cottonwood andsweetgum, which inhabit bottom lands, can be transformed to express merA and merB. These species have roots that grow in the same general areaof the sediment as sulfate-reducing bacteria. As the transgenic plantroots take up methyl mercury, MerB breaks the carbon mercury bond toproduce Hg(II). Hg(II) is a highly reactive metal ion and should end upsequestered in plant tissues bound to various thiol groups.

[0085] Hg(II) produced from the MerD reaction and additional Hg(II)taken up from the environment through its normal mining of nutrients isreduced to Hg(0) by the MerA reaction. Hg(0) is released directly fromthe roots or transpired up the vascular system of the plant, as arewaste gasses like CO₂ from some plants [Dacey, J. W. (1980) Science 210:1017-1019; Dacey, J. W. (1981) Ecology 62:1137; Raven et al. (1986) In:Biology of Plants, Worth Publishers, N.Y., p.775]. By lowering the totallevels in the soil, less methyl mercury will be produced bysulfate-reducing bacteria. Using the MerA and MerB together intransgenic plants at contaminated sites lowers total Hg(II) levels anddestroys environmental methyl mercury, thus preventing a large portionof the methyl mercury from moving through the environment.

[0086] The Hg(0) entering the environment joins the enormous and stablepool of Hg(0) in the atmosphere (Nriagu (1979) In: The Biogeochemistryof Mercury in the Environment, (New York: Elsevier) with half life ofover one year. Because Hg(0) is not easily returned to earth, this poolis not thought to contribute less significantly to manmade contaminationof the environment. In contrast atmospheric Hg(II) species (i.e.,mercury released from coal burning or methyl mercury released naturally)are rapidly returned to earth by rain and dry deposition with ahalf-life of about 1-2 weeks. Thus, volatilization of relatively smallamounts of Hg(0) with good air circulation effectively removes mercuryfrom terrestrial and aquatic environments.

[0087] Once a transgenic plant population expressing MerA and MerB isestablished, these plants efficiently process mercury. Over thesubsequent few decades these plants remove or detoxify most mercury fromat a site. Relying only on currently available biological and chemicalprocessing, the efflux rates of Hg(0) from mercury contaminated sitesare extremely slow. At one such government site it is estimated thatonly 10 kg of the 80,000 kg present in the soil is released as Hg(0) peryear (Lindberg et al. (1995) Environ. Sci. Tech. 29,126-135). The levelsof atmospheric mercury at this and most sites (4-10 ug/m.sup.3) are10,000 fold below what the EPA/OSHA recommend as the maximum allowablelevels (U.S. Public Health Service (1994) Toxicological Profile forMercury. In: Regulations and Advisories, U.S. Public Health Service,Washington, D.C., pp. 261-269). Even if transgenic plants at this siteincreased the efflux rate of metallic mercury 200 times, the level ofatmospheric mercury would still be 50 fold below these allowable levels.The transgenic plants of the present invention allow the efficientremoval of toxic metal compounds such as methyl mercury and ionicmercury from soil, sediment, and aquatic environments, thus meeting alongfelt need for efficient bioremediation of metal and organometalcontaminated sites.

The Operons of the Vector

[0088] The cry2Aa2 Operon. The preferred embodiment of the invention isthe use of Bacillus thuringiensis (Bt) cry2Aa2 operon as a model systemto demonstrate operon expression and crystal formation via thechloroplast genome of tobacco. This operon contains three open readingframes (ORFs). Cry2Aa2 is the distal gene of this operon. The ORFimmediately upstream of cry2Aa2 codes for a putative chaperonin thatfacilitates the folding of cry2Aa2 (and other selected target proteins)to form proteolytically stable cuboidal crystals. Because CRY proteinlevels decrease in plant tissues late in the growing season or underphysiological stress, a more stable protein expressed at high levels inthe chloroplast throughout the growing season should increase toxicityof Bt transgenic plants to target insects and help eliminate thedevelopment of Bt resistance. The function of the third ORF is not yetknown. The invention comprises the operon with the third gene and alsowith the operon without the third gene.

[0089] The mer Operon. Another embodiment of the invention uses the merOperon. The genes for mercury resistance are known as Mer genes, theyare found in operons of bacterial pasmids; different genes constituteoperons, but the two most important are: the merA that codes for themercuric ion reductase and the merB that codes for the organomercuriallyases (Foster, 1983; Summers et al. 1978, 1986). Mer A is a 1.7 kb genethat needs NADPH as a co-factor to reduce mercury to a volatile,non-reactive and less toxic form of mercury (Hg0) (Begley et al. 1986).Mer B is a 638 bp gene that undergoes the protonolysis oforganomercurials by removing the organic group and releasing elementalmercury, which is detoxified by mera (Jackson et al. 1982). Apolycistron containing both genes allows effective degradation ofmercury and organomercurials

Alternative Operons

[0090] Other Cry or Cyt operons may be used in this invention. Anyoperon which comprises at least one of the 133 genes shown in thearticle MMBR, September 1998, pages 805-873, Vol 62, No. 4, Revision ofthe Nomenclature for the BT Insecticidal Crystal Proteins by Crickmon etal., the genes of which codes for the corresponding BT protein; and thechaperonin which facilitates protein folding can be used. Likewise, anyoperon which comprises at least one of the toxins enumerated in Table15.1 or at least one of the of Molecular Biotechnology by Glick andPasternak can be used. Similarly, any operon which comprises a genewhich codes for a delta-endotoxin and the chaperonin which facilitatesprotein folding can be used In addition, any operon which comprises atleast a plasmid identified in Table 13.1 of the of MolecularBiotechnology by Glick and Pasternak can be used.

The Chaperonins

[0091] Chaperonins are a class of a protein referred to as chaperoneswhich has been shown to consist of helper proteins in chain folding andassembly with the cells (Gierasch and King, 1990). They facilitate thefolding and assembly of newly synthesized polypeptide chains intofictional three-dimensional structures by preventing off-pathwayreactions during folding that lead to aggregation (Agashe V R et. al.2000). Chaperonins provide a sequestered environment in which foldingcan proceed unimpaired by intermolecular interactions between non-nativepolypeptides (Agashe V R et. al. 2000). Those skilled in the art will befamiliar with the E. Coli chaperonins: groEL and groES (Viitanen P V et.al. 1995), (Gierasch and King, 1990). Plant chaperonins chaperonin-60and chaperonin-10, which are homologous of gro-EL and gro-ES,respectively. Homologous of the E. Coli groEL and groES continue to beidentified. For instance, a stable complex of the chaperonins has beenisolated and crystallized from the extremely thermophilic bacteriumThermus thermophilus (Lissin N M et. al. 1992). Likewise, plantchaperonins—located both in plastids and the cytosol, continue to beidentified (Baneyx F. et. al., 1995; Viitanen P V et. al., 1995; Burt WJ et. al. 1994, Grellet F. et. al. 1993; Bertsch U et. al., 1992). Thesearticles are hereby incorporated in their entirety by reference.

[0092] The preferred embodiment of this invention use of those bacterialchaperonins that are capable of facilitating the crystallization of theBt endotoxin polypeptides by means of the UV Ct vector and in thetransformed plants.

The Vectors

[0093] This invention contemplates the use of vectors which are capableof stably transforming the chloroplast genome. Such vectors includechloroplast expression vectors such as pUC, pBlueScript, pGEM, and alothers identified by Daniell in U.S. Pat. Nos. 5,693,507 and 5,932,479.These publications and patents are herein incorporated by reference tothe same extent as if each individual publication or patent wasspecifically and individually indicated to be incorporated by reference.

[0094] Universal Vector. A preferred embodiment of this inventionutilizes a universal integration and expression vector competent forstably transforming the chloroplast genome of different plant species(Universal Vector). The universal vector and its construction have beendescribed by the earlier Publication No. WO99/10513, Internationalpublication date: Mar. 4, 1990, which is herein incorporated in itsentirety.

[0095] The 4.0 kb cry2Aa2 operon was inserted into the universalchloroplast expression vector pLD CtV2 (5.8 kb) to form the final E.coli and tobacco shuttle vector pLD-BD Cry2Aa2 operon (9.8 kb) (FIG.1A). This vector could be used to transform chloroplast genomes ofseveral plant species because the flanking sequences are highlyconserved among higher plants. This vector contains the 16S rRNApromoter (Prrn) driving the aadA gene (aminoglycoside3′-adenylyltransferase) for spectinomycin selection and the three genesof the cry2Aa2 operon. The terminator is the psbA 3′ region from thetobacco chloroplast genome from a gene coding for the photosystem IIreaction center component. The 16S rRNA promoter is one of the strongchloroplast promoters recognized by both nuclear and plastid encoded RNApolymerases in tobacco and the psbA 3′ region stabilizes the transcriptof foreign genes. This construct integrates both genes into the spacerregion between the chloroplast transfer RNA genes coding for isoleucineand alanine within the inverted repeat (IR) region of the chloroplastgenome by homologous recombination. The integration into thesetranscribed spacer regions allow the gene to be inserted withoutinterfering with gene coding regions. Also, each genome will contain twogene copies due to integration into the two inverted repeat regionsresulting in a higher copy number (7,000-8,000 copies per cell) andhigher levels of expression. However, the two genes may also beintegrated outside of the IR region resulting in a lower copy number.Chloroplast transgenic plants were obtained as described previously byDaniell (1993, 1997).

[0096] Mer operon vectors with and without terminator: In order tounderstand the role of 3′UTRs in chloroplast foreign gene expression (inmRNA stability and transcription termination), chloroplast vectors withand without 3′ UTRs were made. The PCR products, merA and merB werecloned independently into the pCR2.1 vector (In Vitrogen). Then, theorientation of the merB gene integration was checked. Once the correctorientation was found, the vector TA-merB was cut with a ClaI/EcoRV.MerA was cut with ClaI and EcoRV, and the fragment (merA) was isolatedby gel electrophoresis. Then, the merB and merA genes were ligated.After the ligation, both genes (merB and merA) in-frame were ready forinsertion into the pLD vector. After cutting pLD vector with PstI tolinearize the vector and to remove the terminator, the merB-merAcassette extracted from the TA-vector was ligated into the pLD vector(FIG. 8). The final step was to check for correct orientation. Thus thepLD-merAB vector lacking 3′ UTR was constructed.

[0097] To make the pLD-merA.B-3′UTR, specific set of primers were usedto amplify the whole cassette from the TA-merAB vector. The 5′ primerwas designed with an EcoRV site and the 3′ primer with a XbaI site.These restriction sites allowed the integration into the pLD vector onlyin the right orientation.

Chloroplast Integration of Foreign Genes

[0098] Foreign gene integration into the chloroplast genome wasdetermined by PCR screening of chloroplast transformants (FIG. 1A,B,C).Primers were designed to eliminate spectinomycin mutants and nuclearintegration. The first primer set, 1P1M, lands one primer (1P) on the 3′end of the 16s rRNA flanking sequence and another primer (1M) on aadA(FIG. 1A). This is to distinguish between spectinomycin mutants and truespectinomycin transformants. A 1.6 kb fragment is seen in truetransformants (FIG. 1B, lanes 3,5,6,7). Lane 4 shows a spectinomycinmutant with no PCR product. Untransformed tobacco DNA (lane 2)expectedly shows no product, while pLD-BD cry2Aa2 operon plasmid DNA inlane 8 produced the 1.6 kb fragment. The second primer set, 3P3M, landsone primer (3P) on the native chloroplast genome adjacent to the pointof integration, and another primer (3M) on the aadA gene (FIG. 1A). Thisprimer set generated a 1.65 kb PCR product in chloroplast transformants(FIG. 1C, lanes 3,5,6,7). Untransformed tobacco DNA (lane 2) showed noPCR product, and pLD-BD cry2Aa2 operon plasmid DNA in lane 8 also showedno PCR product because 3P lands on native chloroplast DNA. Lane 4,wasnegative for chloroplast integration and again proving this transformantto be a spectinomycin mutant.

[0099] Southern blot analysis was done to further demonstratesite-specific chloroplast integration of the 4.0 kb cry2Aa2 operon andto determine heteroplasmy or homoplasmy (FIG. 2). Bglll digested DNAfrom transformed plants produce 8.42 kb and 1.4 kb fragments (FIG. 2B)when probed with the 0.81 kb probe (FIG. 2A) that hybridizes to the trnIand trnA flanking sequences. Transgenic plant DNA (T₀ and T₁) producedthe 8.42 kb and 1.4 kb fragments (FIG. 2C, lanes 3-9). A 4.47 kbfragment (FIG. 2A) is seen in untransformed plant DNA (FIG. 2C lane 2).T₀ plant DNA also shows this native untransformed 4.47 kb fragment (FIG.2C, lanes 3-7), thereby showing heteroplasmy in the T₀ generation. This4.47 kb native band is absent from the T₁ generation (FIG. 2C, lanes8-9), thus indicating homoplasmy. If only a fraction of the genomes weretransformed, the gene copy number should be less than 8,000 per cell.Confirmation of homoplasmy in T₁ transgenic lines indicates that theCry2Aa2 operon gene copy number could be as many as 7000-8,000 per cell.

[0100] CRY2Aa2 protein expression and quantification: Expression profileof the operon derived (OD) Cry2Aa2 and single gene derived (SG) Cry2Aa21is shown on a Coomassie stained SDS-PAGE gel (FIG. 3). The primary goalof this experiment is to investigate the location of the operon derivedCry2Aa2 protein (the pellet or supernatant) and correlate with cuboidalcrystals observed in electron micrographs (see FIG. 6). Lane 2 containspartially purified 65 KD Cry2Aa2 from E. coli. Because crystallineCry2Aa2 inclusion bodies are solubilized at high alkaline pH, the 50 mMNaOH solubilized pellet was analyzed from each plant sample aftercentrifugation for 20 min at 13,000 g (lanes 3,5,7). Results show thatOD Cry2Aa2 expression forms crystalline inclusion bodies because theprotein is found mostly in the pellet after centrifugation (lanes 5-6).In contrast, expression of SG Cry2Aa2 is observed in both the pellet andthe supernatant (lanes 3-4). No Cry2Aa2 expression was seen inuntransformed tobacco in either the supernatant or the pellet (lanes 7,8).

[0101] Cry 2Aa2 polypeptides (FIG. 3, lanes 3, 5) were scanned usingStorm 840 Gel Scanner and Image Quant Software Molecular Dynamics). Theoperon-derived expression results only in a 2.5 fold more accumulationof Cry2Aa2 than that of single gene derived cry2Aa2 in the pelletfraction; this does not correlate with more than 100 fold differenceobserved in ELISA (FIG. 4). The reason for this discrepancy is theextreme difference in solubilization between SG Cry2Aa2 derivedamorphous inclusion bodies and the OD Cry2Aa2 derived cuboidal crystals,as reported previously.

[0102] Despite the large difference in protein accumulation (as shown byELISA and electron micrographs, FIGS. 4, 6), the concentration ofsolubilized protein loaded in the pellet fraction was similar in SGCry2Aa2 and OD Cry2Aa2 (FIG. 3, lanes 3, 5). Attempts to completelysolubilize crystalline inclusion bodies for SDS PAGE analysis were notsuccessful because higher pH interfered with gel electrophoresis andrepeated dilution decreased protein concentration below detectablelevels in Coomassie stained gels.

[0103] However, for quantification using ELISA it was possible tocompletely solubilize crystalline inclusion bodies under optimalconditions and dilute the protein to fit within the linear range of theCry2aA2 standard. Therefore, protein expression levels of SG Cry2Aa2 andOD Cry2Aa2 were quantified using ELISA (FIG. 4). Additionally, CRYprotein accumulation in young, mature, and old transgenic leaves derivedfrom a single gene or operon was compared to investigate their stabilityover time. Young, mature, and old leaves expressed SG Cry2Aa2 at 0.014%,0.36%, and 0.03% respectively (FIG. 4A). Cry2Aa2 levels peaked in themature leaf (0.36%) and drastically declined to 0.03% as the plantsenesced. However, young, mature, and old leaves containing OD Cry2Aa2accumulated at 34.9%, 45.3%, and 46.1% respectively (FIG. 413). As thesetransgenic plants aged, OD Cry2Aa2 concentrations remained stable anddid not decline like the SG Cry2Aa2. The presence of theoperon-expressed putative chaperonin should enable the toxin to befolded into stable crystalline structures that are protected fromdegradation. Based on quantitative expression, the cry2Aa2 operonderived expression levels are comparable to that of the RuBisCo, themost abundant protein on earth that compromises up to 65% of leafsoluble protein.

[0104] Insect Bioassays: Five-day-old tobacco budworm (Heliothisvirescens), ten day old cotton bollworm (Helicoverpa zea) and beetarmyworm (Spodoptera exigua) larvae consumed the entire leaf after 24hrs on the untransformed control (FIGS. 5A, D, G). H. virescens feedingon SG Cry2Aa2 leaves died after 5 days (FIG. 5B) while insects diedafter 3 days on OD Cry2Aa2 leaves (FIG. 5C). For SG Cry2Aa2, H. zeaconsumed considerable leaf material after 24 hr, stopped feeding afterthree days and died after five days (FIG. 5E). H. zea consumed verylittle OD Cry2Aa2 material after 24 hours, stopped feeding, and diedafter five days (FIG. 5F). S. exigua feeding on SG Cry2Aa2 (FIG. 5H) orOD Cry2Aa2 (FIG. 51) were lethargic after 24 hours and died after 48hours. Milkweed leaves dusted with OD Cry2A2 transgenic pollen were nottoxic to Monarch butterfly larvae (data not shown) confirming earlierobservations that foreign proteins are not present in tobacco pollen.

[0105] Electron Microscopic Analysis: Untransformed and transgenicleafsections were immunogold-labeled with a Cry2A polyclonal antibody(FIG. 6). FIGS. A-C show developmental OD Cry2Aa2 in chloroplasts inyoung, mature, and old leaves, respectively. In a young green OD Cry2Aa2transgenic leaf (FIG. 6A), labeled Cry2Aa2 occupies a significant amountof the chloroplast, but no crystalline structures are observed. In amature green OD Cry2Aa2 transgenic leaf (FIG. 6B), labeled Cry2Aa2occupies a larger amount of the chloroplast than the younger leaf,resulting in crystals. Theses cuboidal crystals are essentiallyidentical to those expressed in wild-type Cry2Aa2 crystals, orrecombinantly in Bt or E. coli. In an old bleached OD Cry2Aa2 transgenicleaf (FIG. 6C), labeled Cry2Aa2 maintains the crystalline structure andoccupies the highest volume of the chloroplast observed, despite beingbleached and senescent. These findings correlate with OD Cry2Aa2 ELISAresults. In young developing leaves, OD Cry2Aa2 begins accumulation(34.9%), folds Cry2Aa2 into a cuboidal configuration in mature leavesoccupying more cell volume (45.3%), and maintains this cuboidalstructure and volume in old leaves (46.1%). Essentially, as thetransgenic OD Cry2Aa2 plant ages, OD Cry2Aa2 is accumulated, folded andmaintained.

[0106]FIG. 6D is a mature green OD Cry2Aa2 transgenic leaf showingcrystal formation with no immunogold label. This probably occurs becauseas the Cry2Aa2 is folded by the putative chaperonin, epitopes areconcealed thereby decreasing labeling. Crystal formation in FIG. 6Dwould cause the OD Cry2Aa2 to pellet after centrifugation as seen inSDS-PAGE FIG. 3 (lane 5). In EM analysis of mature leaves expressing SGCry2Aa2 (FIG. 6B), protein aggregation is observed, although nocrystalline folding is seen. Cry2Aa2 immunogold labeling occurs in anarea of much lower density than is seen in OD Cry2Aa2 transgenic plantssuggesting lower expression. These results also correlate with ELISA(0.36% in SG Cry2Aa2 in mature leaves). There is no localized antibodyobserved in untransformed tobacco (FIG. 6F).

[0107] Transgenic phenotypes: Phenotypes of OD Cry2Aab2 transgenicplants are not morphologically different from SG Cry2Aa2 transgenicplants (FIG. 7). Therefore, higher levels of expression and accumulationof CRY proteins did not visibly impact their phenotype. Both transgenicplants flowered and set seeds. Characterization of OD Cry2Aa2 T₁transgenic plants for stable integration and transmission of foreigngenes has been shown earlier (FIG. 2).

Possibility of Gene Pyramiding

[0108] This invention enables expression of polycistrons in thechloroplast genome. In contrast to prior efforts in engineering geneexpression in transgenic plants, the present invention allows for thisachievement in a single transformation event that is environmentallysafe. The invention thus opens the possibility for gene pyramiding: theinsertion of multiple insecticidal genes. The invention contemplatesoperons which include not only Bacillus thuingiensis (B.t.) insecticidaltoxin genes, but also non-B.t. insecticidal toxin genes such ascholesteral oxidase, alpha-amylase inhibitors, protease inhibitors, thecowpea trypsin inhibitors, and the potato proteinase inhibitor II.Inclusion of multiple heterologous insecticidal toxin genes retards theability of insects to develop resistance to bio-pesticides.

Expression of Biological Pathways

[0109] Further, this invention provides a method of engineeringbiological pathways into the chloroplast genome in a singletransformation event that is environmentally safe. Because geneexpression is controlled by one promoter, DNA sequences encoding thedifferent genes necessary in a pathway can be co-expressed to the samelevels. Once expressed, the genes of the pathway can act in concert.Gene expression can result in synthesis of enzymes that confer desiredtraits such as degradation of metal ions,herbicides,pesticides,solvents, toulene, napthalene, and otherxenobiotics. An example is the chloroplast transformation of plantchloroplasts with the Mer operon leading to the biodegradation ofmercury and organomercurials. Other pathways include the pigmentbiosynthesis pathway, biosynthetic pathways for enzymes that are couldconfer desired traits such as degradation of xenobiotic compounds notedabove, pathways for amino acids such as the lysine biosynthetic pathway,and pathways for the synthesis of vitamins, carbohydrates, fatty acids,biopolymers and polyesters. Further examples are provided in chapters 12and 13 of Molecular Biotechnology by Glick and Pasternak, which isherein incorporated by reference. Other xenobiotics which can bedegraded using the system of this invention include those given in U.S.Pat. No. 4,259,444 to Chakrabarty which is herein incorporated byreference.

[0110] Expression of pathways can result in the production of compoundssuch as amino acids, fatty acids, carbohydrates, polymers, vitamins,antibiotics and dyes.

Efficient Expression of Bio-pharmaceuticals

[0111] The ability to express polycistrons also opens up the possibilityof efficiently expressing bio-pharmaceuticals such as monoclonalantibodies. Those skilled in the art will know the four DNA sequencesencoding proteins necessary to compose the molecule. Those skilled inthe art will also know that these proteins should be produced in equalamounts (the same stoichiometric ratio). The PCT application entitled“Production of Antibodies in Transgenic Plastids,” filed on Feb. 28,2001 by Daniell, is hereby incorporated by reference to offer examplesof such proteins. This invention allows for the coordinated expressionof these sequences because they are driven by the same promoter. Thismethod avoids the problems of the prior art; namely the pitfalls ofnuclear transformation such as the positional effect and gene silencing.

Application to Other Plants

[0112] This invention provides any higher plants, such asmonocotyledonous and dicotyledonous plant species. The plants that maybe transformed via the universal vector with an antibiotic selectablemarker may be solanacious plants or plants that grow underground. Mostimportantly, this invention is applicable to the major economicallyimportant crops such as maize, rice, soybean, wheat, and cotton. Anon-exclusive list of examples of higher plants which maybe sotransformed include cereals such as barley, corn, oat, rice, and wheat;melons such as cucumber, muskmelon, and watermelon; legumes such asbean, cowpea, pea, peanut; oil crops such as canola and soybean;solanaceous plants such as tobacco; tuber crops such as potato and sweetpotato; and vegetables like tomato, pepper and radish; fruits such aspear, grape, peach, plum, banana, apple and strawberry, fiber crops likethe Gossypium genus such as cotton, flax and hemp; and other plants suchas beet, cotton, coffee, radish, commercial flowing plants, such ascarnation and roses; grasses, such as sugar cane or turfgrass; evergreentrees such as fir, spruce, and pine, and deciduous trees, such as mapleand oak.

[0113] The invention now being generally described, it will be morereadily understood by reference to the following examples which areincluded for purposes of illustration only and are not intended to limitthe present invention.

[0114] In the experimental disclosure which follows, all temperaturesare given in degrees centigrade (.degree), weight are given in grams(g), milligram (mg) or micrograms (.mu.g), concentrations are given asmolar (M), millimolar (mM) or micromolar (.mu.M) and all volumes aregiven in liters (l), milliliters (ml) or microliters (.mu.l), unlessotherwise indicated.

[0115] The invention is exemplified in the following non-limitingexamples which are only for illustrative purposes and are not intendedto limit the scope of the invention.

EXAMPLE 1

[0116] Bombardment and selection of transgenic plants: Tobacco plantswere grown aseptically under fluorescent lights in the laboratory. Seedswere germinated on MSO medium at 27° C. with photoperiods of 16 hourlight and 8 hour dark Microcarriers coated with pLD-BD Cry2Aa2 operonDNA was used to bombard sterile leaves using the Bio-RadPDS-1000/Hebiolistic device as described by Daniell (1997). Bombarded leaves weresubjected to two rounds of selection on RMOP medium containing 500 μg/mlof spectinomycin to regenerate transformants.

[0117] PCR Analysis: DNA was extracted from leaves using the QIAGENDNeasy Plant Mini Kit. PCR was done using the Perkin Elmer Gene Amp PCRSystem 2400. All PCR reactions were performed using the Qiagen Taq DNAPolymerase Kit. Primer sequences used were: IP (5′-ACAATGTAGCCGTACTGGAAGGTGCG GGTG-3′), 1M (5′-CGCGCTT AGC TGGATAACGCCACGGAA-3), 3P(5′-AAAACCCGTCCTCA GTTCGGATTGC-3′), and 3M (5′-CCGCGTTGTITCATCAA GCCTTACG-3′).Samples were run for 30 cycles with the following sequence: 94° C. for 1minute, 70° C. for 1.5 minutes, and 72° C. for 3 minutes. PCR productswere separated on 0.8% agarose gels.

[0118] Southern Blot Analysis: DNA from transformed and untransformedplants was digested with BgIII and transferred to a nylon membrane bycapillary action. The 0.81 kb probe was generated by digesting pLD-CtV2vector DNA with BamHl/BgIII and labeled with ³²P using the ProbeQuant™G-50 Micro Columns (Amersham). Labeled probe was hybridized with thenylon membrane using the Stratagene QUICK-HYB hybridization solution andprotocol.

[0119] SDS-PAGT Analysis: Transgenic and untransformed leaf material(600 mg) was ground to a powder in liquid nitrogen. Protein extractionbuffer from the Cry2Aa2 plate kit from Envirologix (Portland, Me.) usedfor quantification was added to the powder and further grinding wasperformed. The mixture was centrifuged at 4° C. at 13,000 g for 20minutes. The supernatant was removed, boiled in sample buffer, andloaded on a 10% SDS-PAGE gel. The pellet was resuspended in 50 mM NaOHand centrifuged at 4° C. at 5000 g for 5 minutes to pellet cell debris.The supernatant was removed, boiled in sample buffer, and loaded on a10% SDS-PAGE gel at 200V for 4 hours. Gels were stained for 2 hours withR-250 Coomassie Blue and destained overnight in 40% methanol and 10%acetic acid. The DC protein assay by Bio-Rad was used to determine totalsoluble and pellet protein concentration as followed by protocol.

[0120] ELISA: A Cry2Aa2 plate kit from Envirologix was used. Leavesexpressing the SG Cry2Aa2, the OD Cry2Aa2, and untransformed tobaccowere quantified and compared. Approximately 20 mg of leaf was ground in100 μl of 50 mM NaOH to solubilize CRY proteins. Transgenic leafextracts were diluted to fit in the linear range of the provided Cry2aA2standard. The μQuant microtiter plate reader from Bio-Tek read the plateat 450 nanometers (nm). A 1 ppm Cry2Aa2 standard was supplied by the kitand was used in the line arrange between 200-1000 ng for quantification.Color development is proportional to Cry2Aa2 concentration in the sampleextract. The DC protein assay by Bio-Rad was used to determine totalsoluble protein concentration as followed by protocol.

[0121] Insect Bioassays: Leaf disc bioassays were conducted on ca. 2 cm²excised leaf material, and placed on distilled water-soaked cardboardlids in 50×12 mm plastic petri dishes. Insects used were susceptible H.virescens (YDK) obtained from Fred Gould, North Carolina StateUniversity, H. zea obtained from the USDA AIRS facility in Tifton, Ga.and S. exigua from the lab of William Moar. Insects were tested as fiveday or ten day old. All larvae were reared on typical lepidopteranartificial diet prior to use. Two insects were assayed per leaf sample,except H. zea in which only 1 insect was added per leaf sample due tothe cannibalistic nature of the insect (2 leaf samples for H. zea). Allleaf samples for each replicate were from the same leaf. Two sampleswere evaluated per treatment, and observed daily for mortality and leafdamage for 5 days. Treatments were replicated three times.

[0122] Transmission Electron Microscopy and Immunogold Labeling:Immunogold labeled electron microscopy was performed as described by A.J. Vrekleij et al.. Sections were first incubated with 0.05M glycine inPBS buffer (10 mM phosphate buffer, 150 mM NaCl pH 7.4) for 15 minutesto inactivate residual aldehyde groups. The grids were then blocked byplacing them onto drops of PBS with 5% BSA and 0.1% CWFS gelatinsupplemented with 5% normal serum for 30 minutes, washed on drops ofincubation buffer 3 times for 5 min each, and then incubated for 45 minwith the polyclonal Cry2Aa2 to detect tobacco expression (diluted1:10,000 in incubation buffer). To remove unbound primary antibody,sections were washed on drops of incubation buffer 6×5 min each.Sections were then incubated for 2 hours with a goat anti-rabbit IgGsecondary antibody conjugate to 10 nm gold diluted 1:100 in incubationbuffer. Sections were subsequently washed 6×5 minutes in incubationbuffer, 3×5 min with PBS, and post-fixed in 2% glutaraldehyde diluted inPBS for 5 min. Following post-fixation, sections were washed in PBS 3×5minutes, in distilled water 5×2 min each, and post-stained usinguranylacetate and lead citrate. Sections were then examined in a ZeissEM 10 transmission electron microscope at 60 kv.

EXAMPLE 2

[0123]E. Coli Transformants. Due to the similarity of protein syntheticmachinery (Brixey et al. 1997), expression of all metal resistanceconferring chloroplast vectors are first tested in E. coli before theiruse in tobacco transformation The activity of the enzymes, mercury ionreductase (merA) and organomercurial lyase (merB) are tested bytransforming E. coli (XLI-blue) with the recombinant plasmids andgrowing them in LB solid medium with HgCl₂ (FIG. 9). The cells, control(XLI-bue), pLD-merAB and pLDmerAB-3′UTR are grown in differentconcentrations of Hg Cl₂. Control cells do not grow even atconcentrations less than 25 μM Hg Cl₂ but the transformed cells growwell even at 100 μM HgCl₂-(FIG. 10). The ability to grow at these highconcentrations of mercury in which control is not able to grow, confirmsthe functionality of both enzymes. Control and transformed clones aregrown in LB with 500 μg/ml of spectinomycin for 24 hours at 37° C. WhenOD₆₀₀ is measured, 1.247 for the clone with 3′UTR, 0.165 for the clonelacking the 3′UTR, and zero absorbance for the control is observed. Asexpected the pLD-merAB-3′UTR transformed clone shows a higher growthrate probably caused by the 3′ effective termination which allows cellsto make more copies of the mer operon transcript that contain only theaadA, merA and merB genes. In chloroplast genome we expect a minoreffect in the transcription termination efficiency because theterminator of the genes close to the cassette and downstream can serveas a terminator, once it is integrated in the chloroplast genome byhomologous recombination.

[0124] Bombardment and regeneration of chloroplast transgenic plants:Tobacco (Nicotiana tabacum var. Petit Havana) plants are grownaseptically by germination of seeds on MSO medium (Daniell 1993). Fullyexpanded, dark green leaves of about two month old plants are bombardedas described by Daniell (1997). The plants are maintained under 500μg/ml spectinomycin selection in the three phases; first selection (RMOPmedium), second round of selection (RMOP medium) and third selection MSO(rooting medium) (FIG. 10). After these selection events, positivetransformants are transferred to soil (FIG. 10). The plants are testedfor integration of the genes in the chloroplast at first round ofselection and before transplanting them to soil. The use of PCR withspecific primers that land in the chloroplast genome and in the genecassette allows us to eliminate mutants and show integration of theselectable marker gene and the mer genes (FIG. 11). After PCR testing,the plants are grown in soil and the seeds are collected.

[0125] Polymerase Chain Reaction: PCR is done using DNA isolated fromcontrol and transgenic plants to distinguish a) true chloroplasttransformants from mutants and b) chloroplast transformants from nucleartransformants. Primers for testing the presence of the aadA (a gene thatconfers spectinomycin resistance) in transgenic plants are landed on theaadA coding sequence and 16S rRNA gene (primers 1IP & 1M). In order totest chloroplast integration of the mer genes, one primer is landed onthe aadA gene while another is landed on the native chloroplast genome(primers 3P&3M). No PCR product is obtained with nuclear transgenicplants using this set of primers. The primer set (5P & 2M) is used totest integration of the entire gene cassette without any internaldeletion or looping out during homologous recombination, by landing onthe respective recombination sites. This screening is essential toeliminate mutants and nuclear transformants. In order to conduct PCRanalyses in transgenic plants, total DNA from unbombarded and transgenicplants are isolated as described by Edwards et al. (1991). Chloroplasttransgenic plants containing the mer gene are moved to second round ofselection in order to achieve homoplasmy.

[0126] Southern Blot Analysis: Southern blots are done to determine thecopy number of the introduced foreign gene per cell as well as to testhomoplasmy. There are several thousand copies of the chloroplast genomepresent in each plant cell. Therefore, when foreign genes are insertedinto the chloroplast genome, it is possible that some of the chloroplastgenomes have foreign genes integrated while others remain as the wildtype (heteroplasmy). Therefore, in order to ensure that only thetransformed genome exists in cells of transgenic plants (homoplasmy),the selection process is continued. In order to confirm that the wildtype genome does not exist at the end of the selection cycle, total DNAfrom transgenic plants is probed with the chloroplast border (flanking)sequences (the trnI-trnA fragment). If wild type genomes are present(heteroplasmy), the native fragment size will be observed along withtransformed genomes. Presence of a large fragment (due to insertion offoreign genes within the flanking sequences) and absence of the nativesmall fragment should confirm homoplasmy (Daniell et al., 1998; Kota etal., 1999; Guda et al., 2000).

[0127] The copy number of the integrated gene is determined byestablishing homoplasmy for the transgenic chloroplast genome. TobaccoChloroplasts contain 5000-10,000 copies of their genoine per cell(Daniell et al. 1998). If only a fraction of the genomes are actuallytransformed, the copy number, by default, must be less than 10,000. Byestablishing that in the transgenics, the merAB inserted transformedgenome is the only one present, one could establish that the copy numberis 5000-10,000 per cell. This is done by digesting the total DNA with asuitable restriction enzyme and probing with the flanking sequences thatenable homologous recombination into the chloroplast genome. The nativefragment present in the control should be absent in the transgenics. Theabsence of native fragment proves that only the transgenic chloroplastgenome is present in the cell and there is no native, untransformed,chloroplast genome, without the mer genes present. This establishes thehomoplasmic nature of our transformants, simultaneously providing uswith an estimate of 5000-10,000 copies of the foreign genes per cell.

[0128] Northern Blot Analysis: Northern blots are done to test theefficiency of transcription of the merAB operon. Total RNA is isolatedfrom 150 mg of frozen leaves by using the “Rneasy Plant Total RNAIsolation Kit” (Qiagen Inc., Chatsworth, Calif.). RNA (10-40 μg) isdenatured by formaldehyde treatment, separated on a 1.2% agarose gel inthe presence of formaldehyde and transferred to a nitrocellulosemembrane (MSI) as described in Sambrook et al. (1989). Probe DNA (merABgene coding region) is labeled by the random-primed method (Promega)with ³²P-dCTP isotope. The blot is pre-hybridized, hybridized and washedas described above for southern blot analysis. Transcript levels arequantified by the Molecular Analyst Program using the GS-700 ImagingDensitometer (Bio-Rad, Hercules, Calif.).

Plant Bloassays

[0129] Germination/Growth Experiments: Seeds of wild-type (Nicotianatabacum var Petit Havana), transgenic plant pLD-MerAB, and transgenicplant pLD-MerAB-3′UTR are sterilized, vernalized at 4′ C for at least 24h, and germinated on 1% Phytoagar plates (GIBCO/BRL) made with Murashigeand Skoog (4.3 g/liter, GIBCO/BRL) medium containing PMA (phenylmercuricacetate) or mercuric chloride. Seedlings are grown at 22° C. with a 16 hlight/8 h dark period.

[0130] Mercury Vapor Assays: Elemental mercury is relatively insolubleand volatile and lost quickly from cells and media. Volatilized Hg⁰ ismeasured on a Jerome 431 mercury vapor analyzer (Arizona Instrument,Phoeniz, Ariz.) (Rugh, C. L. et al., 1996). Approximately 5-10 seedlings(10-14 day old, 10-25 mg total wet weight) are incubated in 2 ml ofassay medium (50mm Tris.HCL, pH 6.8/50 mM NaCl/25 uM HgCl₂) in a 16×130mm test tube with a side arm for gas removal. The Hg Cl₂ is added toinitiate the assay. The amount of Hg⁰ produced is assayed by bubblingair through the bottom of the sample for 12 sec at 3 cm³/sec andmeasuring the release of Hg⁰. The time zero assay will be takenimmediately after the seedlings are placed in the medium. The sample isthen reassayed every minute for 10 minutes. The volatilized Hg⁰ ismeasured bypassing the air sample released from the side arm directlyover the gold foil membrane resistor of a Jerome 431 mercury vaporanalyzer. The instrument is repeatedly standardized with knownquantities of Hg⁰ (10-200 ng), reduced from HgCl₂ with excess SnCl₂. Theamount of mercury evolved is normalized by dividing the number ofnanograms of Hg⁰ measured by the number of milligrams of seedling tissuein the assay.

[0131] Photosynthetic studies: From transgenic plants and untransformedplants, intact chloroplasts are isolated for photosynthetic studies. O₂evolution is studied in an oxygen evolution electrode in the absence orpresence of different concentrations of HgCl₂ and PMA. Electrontransport is studied with suitable electron donors/acceptors to studyphotosystem I, II or both. PAGE is used to examine the composition ofPSII complex, especially EP33, after incubation of cells or chloroplastsor thylakoid membranes with different concentrations of HgCl₂ and PMA.In vivo chloroplast fluorescence is studied to monitor changes incontrol and transformed cells or chloroplasts to measure Fo, Fm, Fv.

[0132] Inheritance of Introduced Foreign Genes: While it is unlikelythat introduced DNA would move from the chloroplast genome to nucleargenome, it is possible that the gene could get integrated in the nucleargenome during bombardment and remain undetected in Southern analysis.Therefore, in initial tobacco transformants, some are allowed toself-pollinate, whereas others are used in reciprocal crosses withcontrol tobacco (transgenics as female accepters and pollen donors;testing for maternal inheritance). Harvested seeds (T1) are germinatedon media containing spectinomycin. Achievement of homoplasmy and mode ofinheritance is classified by looking at germination results. Homoplasmyis indicated by totally green seedlings (Daniell et al., 1998) whileheteroplasmy is displayed by variegated leaves (lack of pigmentation,Svab & -Maliga, 1993). Lack of variation in chlorophyll pigmentationamong progeny also underscores the absence of position effect, anartifact of nuclear transformation. Maternal inheritance is demonstratedby sole transmission of introduced genes via seed generated ontransgenic plants, regardless of pollen source (green seedlings onselective media). When transgenic pollen is used for pollination ofcontrol plants, resultant progeny would not contain resistance tochemical in selective media (will appear bleached; Svab and Maliga,1993). Molecular analyses confirms transmission and expression ofintroduced genes, and T2 seed are generated from those confirmed plantsby the analyses described above.

EXAMPLE 3

[0133]Chlorella vulgaris transformation vector: The region 16S to 23S ofthe Chlorella vulgaris chloroplast genome is amplified by PCR usingspecific primers complementary to rrn16 and to rrn23. The PCR productwill be cloned into pCR 2.1 vector available from Promega. The PCRproduct 16S to 23S is removed from the pCR2.1 vector by a blunt endrestriction endonuclease and cloned into the pUC19 in which the multiplecloning site has been removed using a blunt end restriction enzyme(Pvu1I). Then the cassette containing the promoter, the antibioticresistance gene and the merAB genes is inserted into the new vector(Chlorella transformation vector) using a blunt end restriction enzyme(HincII) that is present in the spacer region between trnA and trnT. Thefinal construct is used for the transformation of Chlorella vulgaris(FIG. 12).

[0134] Bombardment and transformation of Chlorella vulgaris: Thebiolistic transformation method (Sanford et al. 1993) is optimized fortransformation of Chlorella vulgarism. Chlorella is grown in liquidheterotrophic medium (5 sporulation agar) at 25° C. to late-log phase(˜6×10⁶ cells/ml). To prepare a monolayer for bombardment (2×10⁷), cellsare collected onto prewetted 45 mm GVWP filters (Millipore) under gentle(30 mBar) vacuum. Gold particles are coated with the transformingplasmid. (Sanford et al. 1993) The monolayer filters are bombarded.Immediately after bombardment, filters are transferred to selectivesolid media containing 500 μg/ml spectinomycin and incubated at 22° C.under high light. After approximately 6 weeks, green colonies are pickedfrom a background of bleached cells onto selective plates and grown foran additional 1-2 weeks. Colonies are harvested and screened forintegration of foreign genes when they reached a diameter ofapproximately 5 mm.

[0135]Chlorella vulgaris Bioassays: Growth and colonies formationbioassay are performed as explained in the plant germination growthexperiment; the only change is the use of Chlorella specific media.

[0136] Mercury vapor assays: Mercury vapor assays are performed in theway explained for plants above except changing the growth media specificto Chlorella, including temperature and light intensity.

[0137] Photosynthetic studies: Photosynthetic studies are performed inthe way explained for plants above except untransformed and transformedChlorella cells will be directly used to study the effect of mercurytoxicity.

EXAMPLE 4

[0138] Synechocystis transformation vector: The region 16S to 23S of theSynechocystis genome is amplified by PCR using specific primerscomplementary to rrn 116 and to rrn23. The PCR product is cloned intothe pCR 2.1 vector available from Promega. The PCR product 16S to 23S isremoved from the pCR2.1 vector by a blunt end restriction endonucleaseand cloned into pUC19 in which the multiple cloning site has beenremoved using a blunt end restriction enzyme (Pvu1I). Then the cassettecontaining the promoter, the antibiotic resistance gene and the merABgenes is inserted into the new vector (Synechocystis transformationvector) using a blunt end restriction enzyme (HincII) that is present inthe spacer region between trnI and trnA. The final construct will beused for the transformation of Synechocystis (FIG. 13).

[0139] Transformation of Synechocystis: A fresh culture of wild type inBG-11 (heterotrophic medium) plus glucose is grown to OD₇₃₀=0.5 after2-3 days of culture. Cells are spun down in sterile 50 ml tubes at roomtemperature and resuspended in the original growth medium to OD₇₃₀=2.5.Transforming DNA is added to resuspended cells in sterile glass culturetubes. Tubes are placed in rack in the growth chamber at 30° C. for 6hours and shaken for 3 hours. Cells (200 μl) are plated on a sterilefilter that has been placed on a BG-11 plus glucose plate and spreadaround. After growth for 24 hours and they are transferred to filters onappropriate medium containing spectinomycin or mercuric chloride.

[0140] Synechocystis Bioassays: Growth and colonies formation bioassayare performed as explained in the plant germination-growth experiment;the only change is the use Synechocystis growth media.

[0141] Mercury vapor assays: Mercury vapor assays are performed in theway explained for plants above except changing the growth media specificto the Synechocystis, including temperature and light intensity.

[0142] Photosynthetic studies: Photosynthetic studies are performed inthe way explained for plants above except untransformed and transformedSynechocystis cells are directly used to study the effect of mercurytoxicity.

EXAMPLE 5

[0143] Lemna transformation vector: The Lemna chloroplast vector, asshown in FIG. 14, is constructed in the same way as explained above fortobacco, with the exception that the Lemna chloroplast DNA flankingsequences are used.

[0144] Bombardment and regeneration of transgenic plants: Lemna plantsare transformed and regenerated in the way explained for tobacco inExample 1 above.

[0145] Plant Bioassays: Various plant bioassays are performed asexplained for tobacco in Example 1 above.

EXAMPLE 6

[0146] Sugarcane transformation vector: The Sugarcane chloroplastvector, as shown in FIG. 15, is constructed in the same way as explainedabove for tobacco, with the exception that the Sugarcane chloroplast DNAflanking sequences are used.

[0147] Bombardment and regeneration of transgenic plants: Sugarcaneplants are transformed and regenerated in the way explained for tobaccoin Example 1 above.

[0148] Plant Bioassays: Various plant bioassays are performed asexplained for tobacco in Example 1 above.

References

[0149] Begley T P A, Walts A E, Walsh Conn. (1986) Mechanistic studiesof a protonolytic organomercurial cleaving enzyme: bacterialorganomercurial lyase. Biochemistry 25: 7192-7200.

[0150] Bernier M, Popovic R, Carpentier R (1993) Mercury inhibition ofphotosystem 11. FBS Lett. 32:19-23.

[0151] Bernier M, Carpentier R (1995) The action of mercury on thebinding of extrinsic polypeptides associated with water oxidizingcomplex of photosystem II.

[0152] Bizily S, Rugh C C, Summers A O, Meagher R B (1999)Phytoremediation of methyl mercury pollution: merB expression inArabidopsis thaliana plants confer resistance to organomercurial. PNAS96: 6808-6813.

[0153] Bizily S, Rugh C C, Meagher R B (2000) Phytoremediation ofhazardous organomercurials by genetically engineered plants. NatureBiotechnology 18; 213-217.

[0154] Bogorad, L. Engineering chloroplasts: an alternative site forforeign genes, proteins, reactions, and products. Trends inBiotechnology. 18, 257-263 (2000).

[0155] Bradley, D. et al. The insecticidal CrylB crystal protein ofBacillus thuringiensis ssp. Thuringiensis has dual specificity tocoleopteran and lepidopteran larvae. J. Invert. Pathol. 65, 162-173(1995).

[0156] Carlson P S (1973) The use of protoplasts for genetic research.Proc. Natl. Acad. Sci. USA 70:.598-602.

[0157] Compeau G C, Bartha R (1985) Sulfate-reducing bacteria: principalmethylators in freshwater sediments. Appl. Environ. Microbiol, 50:498-502.

[0158] Crickmore, N. & Ellar, D. Involvement of a possible chaperonin inthe efficient expression of a cloned crylIA 8-enclotoxin gene inBacillus thuringiensis. MoL Microbiol. 6, 1533-1537 (1992).

[0159] Crickmore, N., Wheeler, V. & Ellar, D. Use of an operon fusion toinduce expression and crystallisation of a Bacillus thuringiensis5-endotoxin encoded by a cryptic gene. MoL Gen. Genet 242, 365-368(1994).

[0160] Daniell, H. Foreign gene expression in chloroplasts of higherplants mediated by tungsten particle bombardment. Methods Enzymo/. 217,536-556 (1993).

[0161] Daniell, H. et al. Engineering plants for stress tolerance viaorganelle genomes. NATO ASI Series. 86, 589-592 (1994).

[0162] Daniell, H. Transformation and foreign gene expression in plantsmediated by microprojectile bombardment. Meth. Mol. Biol.62,463-489(1997).

[0163] 1 A operons on the formation of Cry2A inclusions in Bacillusthuringiensis. FEMS Microbiol. Lett. 165, 35-41 (1998).

[0164] Daniell, H. et al. Containment of a herbicide resistance throughgenetic engineering of the chloroplast genome. Nature Biotechnol. 16,345-348 (1998).

[0165] Daniell, H. GM crops: public perception and scientific solutions.Trends in Plant Science. 4, 467-469 (1999).

[0166] Daniell, H. New tools for chloroplast genetic engineering. Nat.Biotechno'll. 17, 855-856 (1999).

[0167] Daniell H, Kulandaivelu G and Chandrasingh U (1980) Substitutedp-benzoquinones having high electron affinity as photosystem II electronacceptors. Z. Naturforsch. 35C, 136-138.

[0168] Daniell H and Sarojini G (1981) Site of action of2,5-dimethoxy-3,6-dichloro-p-benzoqliinone in the photosyntheticelectron transport chain. Z. Naturforsch. 36C, 656-661.

[0169] Daniell H, Rarnanujan P, Krishnan M, Gnanam A, Rebeiz C A (1983)In vitro synthesis of photosynthetic membranes: L Development ofphotosystem I activity and cyclic phosphorylation. Biochem. Biophys.Res. Comun. 11 1: 740-749.

[0170] Daniell H, Krishnan M, Renganathan M and Gnanam A (1984)Radioisotopic evidence for the polypeptides associated with photosystemII activity. Biochem. Biophys. Res. Commun. 125,988-995.

[0171] Daniell H, Anbudurai P R, Periyanan S, Renganathan M, Bhardwaj R,Kulandaivelu G and Gnanam A(1985) Oxygenic photoreduction of methylviologen without the involvement of photosystem I during plastiddevelopment. Biochem. Biophys. Res. Commun. 126, 1114-1121.

[0172] Daniell H and McFadden B A (1986) Characterization of DNA uptakeby the cyanobacterium Anacystis nidulans. Mol. Gen. Genetics 204,243-248.

[0173] Daniell H, Sarojini G and McFadden B A (1986) Transformation ofthe cyanobacterium Anacystis nidulans 6301 with the Escherichia coliplasmid pBR322. Proc. Natl. Acad. Sci. USA 83, 2546-2550.

[0174] Daniell H and McFadden B A (1987) Uptake and expression ofbacterial and cyanobacterial genes by isolated cucumber etioplasts.Proc. Natl. Acad. Sci. USA 84: 6349-6353. Daniell H and McFadden B A(1988) Genetic Engineering of plant chloroplasts. U.S. Pat. Nos.5,932,479; 5,693,507.

[0175] Daniell H, Vivekananda J, Neilsen B, Ye G N, Tewari K K, SanfordJ C (1990) Transient foreign gene expression in chloroplasts of culturedtobacco cells following biolistic delivery of chloroplast vectors. Proc.Natl. Acad. Sci. USA 87: 8892.

[0176] Daniell H, Krishnan M, McFadden B A (1991) Expression ofB-glucuronidase gene in different cellular compartments followingbiolistic delivery of foreign DNA in to wheat leaves and calli. PlantCell Reports 9: 615-619.

[0177] Daniell H, Muthukumar B and Lee S B (2001) Marker free transgenicplants: engineering the chloroplast genome without the use of antibioticselection. Current Genetics. In press.

[0178] DeCosa B, Moar W, Lee S B, Miller M, Daniell H (2001).Hyper-expression of the Bt Cry2Aa2 operon in chloroplasts leads toformation of insecticidal crystals. Nature Biotechnol. 19: 71-74.

[0179] De Wilte, C. et al. (2000). Plants as Bioreacters for ProteinProduction: Avoiding the Problem of Transgene Silencing. Plant MolecularBiology 43:347-389.

[0180] Edwards K, Johnstone C, Thompson C (1991) A simple and rapidmethod for preparation of plant genomic DNA for PCR analysis. NucleicAcids Res. 19: 1349.

[0181] Foster T J (1983) Plasmid-determined resistance to antimicrobialdrugs and toxic metal ions in bacteria. Microbiol. Rev. 47: 361-409.

[0182] Ge, B. et al. Differential effects of helper proteins encoded bythe cry2A and cryl Greenplate, J. Quantification of Bacillusthuringiensis insect control protein CrylAc over time in bollgard cottonfruit and terminals. J. Econ. Entomo/. 92, 1377-1383 (1999).

[0183] Gilmour C C, Henry E A, Mitchel R (1992) Sulfate stimulation ofmercury methylation in freshwater sediments. Environ. Sci. Technol. 26:2281-2287.

[0184] Guda, C., Lee, S. B. & Daniell, H. Stable expression of abiodegradable protein-based polymer in tobacco chloroplasts. Plant CellReports. 19, 257-262 (2000).

[0185] Harada M, Minarnata Disease Research Group (1995) Minamatadisease: methylmercury poisoning in Japan caused by environmentalpollution. Crit. Rev. Toxicol. 25: 1-24.

[0186] Kota, et al. Overexpression of the Bacillus thurin iensis (13t)Cry2Aa? protein 9 in chloroplasts confers resistance to plants againstsusceptible and Bt-resistant insects. Proc. Nati. Acad. Sci.96,1840-1845 (1999).

[0187] Kulandaivelu G and Daniell H. (1980) Dichlorophenyl dimethylurea(DCMU) induced increase in chlorophyll a fluorescence intensity: Anindex of photosynthetic oxygen evolution in leaves, chloroplasts andalgae. Physiol. Plant. 48, 385-388.

[0188] Ma, J. et al. Generation and assembly of secretary antibodies inplants. Science. 268, 716-719 (1995).

[0189] McBride K E, Svab Z, Schaaf D J, Hogen P S, Stalker D M, Maliga P(1995) Amplification of a chimeric Bacillus gene in chloroplasts leadsto extraordinary level of an insecticidal protein in tobacco.Bio/technology 13; 362-365.

[0190] Minamata Disiase Research Group (1968)Minamata Disease. (MedicalSchool of Kumamoto University, Kumamoto, Japan)

[0191] Moar, W. et al. Insecticidal activity of the CryllA protein fromthe NRD-12 isolate of Bacillus thuringiensis subsp. kurstaki expressedin Escherichia coli and Bacillus thuringiensis and in a leaf-colonizingstrain of Bacillus cereus. Appl. Environ. Microbiol. 60, 896-902 (1994).

[0192] Moar, W. et al. Development of Bacillus thuringiensis Cryl Cresistance by Spodoptera exigua. Appl. Environ. Microbiol. 61, 2086-2092(1995).

[0193] Navrath, C., Poirier, Y., & Somerville, C. Targeting of thepolyhydroxybutyrate biosynthetic pathway to the plastis of Arabidopsisthaliana results in high levels of polymer accumulation. Proc. Natl.Acad. Sci. 91, 12760-12764 (1994).

[0194] Peerenboom, E (2000) German health minister calls time out for Btmaize. Nature Biotechnol. 18: 374.

[0195] Puchta H (2000) Removing selectable marker genes: taking theshortcut. Trends in plant Science 5:273-274.

[0196] Rashid A, Popovic R (1990) Protective role of CaCl₂ against Pb²⁺inhibition in photosystem IL FEBS Lett. 271: 181-184.

[0197] Roy, H. & Nierzwicki-Bauer, S. RuBisCo: genes, structure,assembly and evolution. The Photosynthetic Apparatus, L. Bogorad, 1.Vasil (eds),pp 347-364, Academic Press, NY. (1994).

[0198] Rugh C C, Summers A O, Meagher R B (1996) Mercuric ion reductaseand resistance in transgenic Arabidopsis thaliana plants expressingmodified bacterial merA gene. PNAS 93: 3182-3187.

[0199] Salt D E et al. (1998) Phytoremediation. Rev. Plant. Physiol.Plant. Mol. Biol. 49-643-668.

[0200] Sanford J C, Smith F D, Russell J A (1993) Optimizing thebiolistic process for different biological applications. MethodsEnzymol. 217: 483-509.

[0201] Sambrook J, Fritch E F, Maniatis T (1989) Molecular cloning. ColdSpring Harbor Press, Cold Spring Harbor, N.Y.

[0202] Sarojini G, Daniell H and Vermaas W F J (1981) Site of electronacceptance by 3,6,dichloro-2,5-dimethoxy-p-benzoquinone and its relationto the bicarbonate effect on photosynthetic electron transport Biochem.Biophys. Res. Commun. 102, 944-951.

[0203] Sidorov V A, Kasten D, Pang S Z, Hajdukiewicz P T J, Staub J M,Nehra N S (1999) Stable chloroplast transformation in potato: use ofgreen fluorescent protein as a plastid marker. Plant Journal 19:209-216.

[0204] Staub J M, Garcia B, Graves J, Hajdukiewicz P T J, Hunter P,Nehra N, Paradkar V, Schlittler M, Caroll J A, Spatola I, Ward D, Ye G,Russell D (2000) High-yield production of human therapeutic protein intobacco chloroplast. Nature Biotechnol. 18: 333-338.

[0205] Summers A O, Silver S (1978) Microbial transformation of metals.Annu. Rev. Microbiol. 32:637-672.

[0206] Summers A O (1986) Organization, expression, and evolution ofgenes for mercury resistance. Annu. Rev. Microbiol 40: 607-634.

[0207] Svab Z, Maliga P (1993) High frequency plastid transformation intobacco by selection for a chimeric aadA gene. Proc. Natl. Acad. Sci.USA 90: 913-917.

[0208] Trebs A (1980) Inhibitors in electron flow: tools for thefunctional and structural localization of carriers and energyconservation sites.

[0209] A. J Vrekleij, J. M. Leunissen (eds.), Immuno-gold Labeling inCell Biology CRC Press, Boca Raton, Fla. (1989).

[0210] Yamamoto, T. & lizka, T. Two types of entomocidal toxins in theparasporal crystals of Bacillus thuringiensis var. kurstaki. ArchivesBiochem. Biophys. 227, 233241 (1983).

[0211] Ye, X et al, Engineering the provitamin A (0-carotene)bipsynthetic pathway into (carotenoid-free) rice enclosperm. Science.287, 303-305 (2000).

[0212] Herbicide Resistance Crops, Agricultural, Environmental,Economic, Regulatory and Technical Aspects, Duke, S. O., edt., CRCPress, Inc. (1996).

[0213] Herbicide Resistance in Plants, Biology and Biochemistry, Powles,S. B., and Holtum, J A M., eds., CRC Press, Inc. (1994).

[0214] Peptides: Design, Synthesis, and Biological Activity, Basava, Cand Anantharmaiah, G. M., eds., Birkhauser Boston, 1994.

[0215] Protein Folding: Deciphering the Second Half of the Genetic Code,Gierasch, L. M., and King, J., eds., American Association For theAdvancement of Science (1990).

1 6 1 30 DNA Artificial Sequence Description of Artificial SequencePrimer 1 acaatgtagc cgtactggaa ggtgcgggtg 30 2 27 DNA ArtificialSequence Description of Artificial Sequence Primer 2 cgcgcttagctggataacgc cacggaa 27 3 25 DNA Artificial Sequence Description ofArtificial Sequence Primer 3 aaaacccgtc ctcagttcgg attgc 25 4 25 DNAArtificial Sequence Description of Artificial Sequence Primer 4ccgcgttgtt tcatcaagcc ttacg 25 5 24 DNA Artificial Sequence Descriptionof Artificial Sequence Primer 5 ctgtagaagt caccattgtt gtgc 24 6 25 DNAArtificial Sequence Description of Artificial Sequence Primer 6tgactgccca acctgagagc ggaca 25

1. A stable chloroplast transformation and expression vector which iscapable of introducing multiple genes into a selected plant by a singleintegration event, wherein each step of said multiple genes is carriedout by an enzyme encoding a heterologous DNA sequence which comprises anexpression cassette, comprising as operably linked components, in the 5′to the 3′ direction of translation, a promoter operative in saidplastids which drives a multi-gene operon, a selectable marker sequence,the multi-gene operon which is functional to co-express multiple enzymesin the plastids, a transcription termination region functional in saidplastids, and flanking each side of the expression cassette, flankingDNA sequences which are homomlogous to DNA sequences inclusive of aspacer sequence off the target plastid genome, whereby stableintegration of the heterologous coding sequence into the chloroplastgenome of the target plant is facilitated throughout homologousrecombination of the flanking sequence with the homologous sequences inthe target plastid gene.
 2. A vector of claim 1, wherein a gene of theoperon codes for an insecticidal toxin crystal protein.
 3. A vector. ofclaim 2, wherein the insecticidal toxin crystal protein is a Bacillusthumngensis (Bt) crystal protein.
 4. A vector of claim 3, whereinanother gene of the operon codes for another insecticidal crystalprotein with a different mode of action
 5. A vector of claim 4, whereinthe multi-gene operon is functional to co-express, in addition to a Btinsecticidal toxin gene, a non-Bt insecticidal toxin gene selected fromat least one of the group of cholesterol oxidase, alpha-amylateinhibitors, protease inhibitors, cowpea trypsin inhibitors and thepotato proteinase inhibitor II, whereby the “pyramiding” of the toxinproduct tends to retard the ability of insects to adapt to theinsecticidal effect of the transgenic target plants.
 6. A vector ofclaim 4 or 5, wherein a second gene of the operon codes for a putativechaperonin which facilitates the folding of the Bt crystal toxin proteinto form proteotically stable cuboidal crystals.
 7. A vector of claim 3,wherein the operon includes one of the 133 genes shown in the articleMMBR, (September, 1998, pages 805-873, Vol 62, No. 4, Revision of theNomenclature for the Br-Pesticidal (insecticidal) Crystal Proteins byCrickmon, et al.), wherein at least one of the genes of the operon codesfor a Bt insecticidal crystal protein and another gene codes for aputative corresponding chaperonin which facilitates the folding and thecorresponding chaperonin facilitates the folding of the Bt protein toproteolytically stable cuboidal crystals shown in that MMBR article. 8.A vector of claim 1, wherein at least one of the gene of the operoncodes for a biopharmaceutical protein.
 9. A vector of claim 7, whereinanother gene of the operon codes for a putative chaperonin whichfacilitates the folding of the protein.
 10. A vector of claim 8, whereinthe protein is insulin or human albumin.
 11. A vector of claim 7,wherein another gene of the operon codes for another biopharmaceuticalprotein other than the gene which codes for the putative chaperonin,which protein is expressed in stoichimetric ratio.
 12. A vector of claim10, wherein the genes of the operon, other than the gene which codes forthe putative chaperonin, codes for biopharmaceutical proteins which areexpressed in stoichimetric ratio.
 13. A vector of claims 7,8,9 or 10which comprises collecting the protein product in a foldedconfiguration, thereby enhancing their stability, and facilitatingsingle step purification.
 14. A method of combating insects whichcomprises applying to the insects or their habitat an insecticidallyamount of the insecticidal crystal protein of claim
 4. 15. A method oftransforming a chloroplast of a selected plant species or the progenythereof to confer insect resistance and producing on a large-scaleforeign protein, said method comprising the steps of: stablytransforming the chloroplast of selected plant cells to express at leastone insecticidal toxin protein and a chaperonin, growing the transformedplant cells under conditions which allow the expression of saidinsecticidal toxin protein and chaperonin.
 16. The method of claim 14,further comprising the steps of culturing said plant cells in a plantgrowth medium comprising spectinomycin, and selecting transformed plantcells capable of growth in the presence of said spectinomycin.
 17. Themethod of claim 15, further comprising regenerating a transformed plantfrom said transformed plant cells.
 18. A transformed plant which hasbeen transformed by the method of any one of claims 14-16.
 19. Thetransformed plant of claim 18, wherein said plant contains a highaccumulation of insecticidal toxin proteins in said plant's leaves,including mature and old bleached leaves.
 20. The progeny, includingseeds, of the transformed plant of claim
 18. 21. A vector of claim 1,wherein the biosynthetic pathway is a bioremediation system thatfunctions to degrade inorganic and organic metal compounds incontaminated sites.
 22. A vector of claim 21, wherein the expressioncassette does not contain a terminator.
 23. A vector of claim 21 orclaim 22, wherein the operon contains the merry resistance codingsequences encoding enzymes Mer A and Mer B.
 24. The vector of claim 23,wherein the bioremediation pathway is driven by a single promoter. 25.The chloroplast transformation and expression vector of claim 24,wherein enzymes of the bioremediation pathway are expressed instoichiometric amounts.
 26. A vector of claim 25, wherein the inorganiccompounds are selected from at least one of the group consisting ofdivalent cations of mercury, nickel, cobalt, trivalent cations of gold,and monovalent cations of silver.
 27. A vector of claim 25, wherein theorganic compounds are selected from at least one of the group consistingof alkyl mercury, alkenyl mercury, alkynyl mercury, aromatic mercurycompounds, alkyl lead compounds, alkyl arsenic compounds and alkylcadmium compounds.
 28. A method of transforming a chloroplast of aselected plant species or the progeny thereof to confer greaterresistance to metal ions than the corresponding parental plant whichdoes not require several back crosses to create complete pathway thatdetoxifies mercury and organiomercurial, said method comprising thesteps of: stably transforming the chloroplast of a plant by inserting anexpression cassette containing the mercury resistance coding sequencesof claim 21 into a plant species or the progeny thereof, growing thetransforming plant species under conditions which allow the expressionof said expression cassette.
 29. The method of claim 28, furthercomprising culturing said plant in a plant growth medium comprising aselector for the corresponding selectable marker of claim 1, andselecting transformed plant cells capable of growth in the presence ofsaid selector.
 30. The method of claim 29, further comprisingregenerating a transformed plant from said transformed plant cells. 31.A stably transformed plant which has been transformed by the methods ofany one of claims 28-30.
 32. The progeny, including seeds, of the stablytransformed plant of claim
 31. 33. A method of phytoremediation ofmercury and organomercurials in soil and ground water, said methodcomprising the steps of: planting the stably transformed plant of eitherclaim 31 or claim 32 in soil contaminated with mercury andorganomercurials and allowing said plants to grow.
 34. A method ofphytoremediation which does not require several back crosses to createcomplete pathway that detoxifies mercury and organomercurial, saidmethod comprising the methods of claims 28-30.
 35. The plants of claim33, wherein the plant contains, products of the bioremediation pathway.36. The products of the stably transformed plant of either claim 31 orclaim 32, wherein said products are metals that are reduced by theenzymes of the bioremediation pathway.
 37. The vector of claim 23 whichis capable of introducing a multiple-step biosynthetic pathway into aselected photosynthetic cell by a single integration event
 38. Thevector of claim 37, wherein the biosynthetic pathway degrades inorganicand organic mercury compounds.
 39. A vector of claim 38, wherein thebioremediation pathway is driven by a single promoter.
 40. A vector ofclaim 38, wherein the enzymes of the bioremediation pathway areexpressed in stoichiometric amounts.
 41. A vector of claim 38, whereinthe inorganic compounds are selected from at least one of a groupconsisting of divalent cations of mercury, nickel, cobalt, trivalentcations of gold, and monovalent cations of silver.
 42. A vector of claim38, wherein the organic compounds are selected from at least one of agroup consisting of alkyl mercury, alkenyl mercury, alkynyl mercury,aromatic mercury compounds, alkyl lead compounds, alkyl arseniccompounds and alkyl cadmium compounds.
 43. A photosynthetic organismtransformed with the vector of claim 38 which is useful forbioremediation of mercury and organomercurial compounds fromcontaminated water bodies.
 44. A method of transforming a chloroplast ofa selected photosynthetic organism to confer greater resistance to metalions, said method comprising the steps of: stably transforming thechloroplast of a photosynthetic organism with the vector of claim 38,growing the transformed photosynthetic organism under conditions whichallow the expression of said expression cassette.
 45. The method ofclaim 44, further comprising culturing said photosynthetic organism in agrowth medium comprising a selector, and selecting transformed cellscapable of growth in the presence of said selector.
 46. The method ofclaim 45, further comprising regenerating a transgenic photosyntheticorganism from said transformed cells.
 47. A method of phytoremediationof mercury and organomercurials in bodies of contaminated water, saidmethod comprising the steps of: treating water contaminated with mercuryand organomercurials with the transgenic photosynthetic organism ofclaim 42 before releasing the water into the environment.
 48. Thephotosynthetic organism of claim 43, wherein said photosyntheticorganism is either a green algae or a cyanobacteria.
 49. Thephotosynthetic organism of claim 48, wherein the green algae isChlorella vulgaris.
 50. The photosynthetic organism of claim 48, whereinthe cyanobacteria is Synechocytis.
 51. A vector of claim 1 wherein amulti-gene operon codes for a protein.
 52. A vector of claim 51, whereinthe protein is a biopharmaceutical protein.
 53. A vector of claim 52,wherein the biopharmaceutical protein is a monoclonal antibody.
 54. Avector of claim 53, wherein the protein is produced in the samestoichiometric ratio.
 55. A vector of claim 4, wherein said another geneof the operon is selected from the group of cholesterol oxidase,alpha-amylase inhibitors, and proteinase inhibitors.
 56. The vector ofanyone of claims 1-13, 21-27, 37-42, 51-55, wherein the promoter is aone functional in green or non-green plastids.
 57. The promoter of claim56, wherein said promoter is selected from the group of psbA, accD, or16srRNA promoters.
 58. The biosynthetic pathway of claim 1, wherein saidbiosynthetic pathway result in the production of compounds such as aminoacids, fatty acids, carbohydrates, polymers, vitamins, antibiotics anddyes.
 59. A vector of claim 8, where the protein is human serum albumin.60. A vector of claim 1, which further comprises flanking each side ofthe expression cassette, flanking DNA sequences which are homologous toa DNA sequence inclusive of a spacer sequence of the target chloroplastgenome, which sequence is conserved in the chloroplast genome ofdifferent plant species, whereby stable integration of the heterologouscoding sequence into the chloroplast genome of the target plant isfacilitated through homologous recombination of the flanking sequenceswith the homologous sequences in the target chloroplast genome.